NaK-ATPase-Derived Peptide SRC Inhibitors and Ouabain Antagonists and Uses Thereof

ABSTRACT

A novel Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades and uses thereof are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/122,205 filed Dec. 12, 2008, the disclosure of which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under the NationalInstitutes of Health (NIH) Grant HL-36573 awarded by the National Heart,Lung and Blood Institute, and NIH Grant GM-78565 awarded by the NationalInstitute of General Medical Sciences. The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 14, 2009, is named420_(—)50445_SEQ_LIST_D2009-07.txt, and is 4 kb in size.

TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION

The present invention is based, in part, on the discovery of aNa/K-ATPase/Src receptor complex, and its uses to develop novel agonistsor antagonists of the receptor. The receptor function of Na/K-ATPase/Srccomplex is altered in many diseases including cancer, tissue fibrosis,congestive heart failure and ischemia/reperfusion injury.

BACKGROUND

The Na/K-ATPase enzyme is ubiquitously expressed in most eukaryoticcells and is essential for maintaining the trans-membrane ion gradientby pumping Na⁺ out and K⁺ into cells (1). Structurally, the enzymeconsists of two non-covalently linked α and β subunits. Similar to otherP-ATPases, including the gastric H/K-ATPase and sarcoplasmic reticulumCa-ATPase (SERCA), the Na/K-ATPase α subunit has 10 transmembranedomains with both N- and C-termini located in the cytoplasm (2,3). Basedon the published crystal structures of both Na/K-ATPase and SERCA, the αsubunit consists of several well-characterized domains: actuator (A)domain consists of the N-terminus and the second cytosolic domain (CD2)connected to transmembrane helices M2 and M3; highly conserveddiscontinuous phosphorylation (P) domain is close to the plasmamembrane; and a relatively isolated nucleotide-binding (N) domain (4).These structures also show a significant movement of A and N domainduring the ion pumping cycle (5,6). It appears that the A domain rotateswhile the N domain closes up during the transport cycle, which opens(E1) and closes (E2) the A, N and P domains. Interestingly, thesedomains have also been implicated in interacting with many proteinpartners, including inositol 1,4,5-trisphosphate (IP3) receptors(IP3Rs), phosphoinositide 3′ kinase (PI3K), phospholipase C-γ (PLC-γ),ankyrin, and cofilin (7-12).

Previously, the inventors and others have demonstrated that binding ofcardiotonic steroids (CTS) such as ouabain to the Na/K-ATPase stimulatesmultiple protein kinase cascades (13,14). Moreover, knockout of Srcabolishes most of these activations (10,15,16). Src, a member of Srcfamily non-receptor kinases, plays an important role in the signaltransduction pathways of many extracellular stimuli, i.e., cytokines,growth factors and stress responses (17) and has been considered as apromising target for therapeutic interventions in certain cancers (18)and bone diseases (19). Several endogenous inhibitors of Src have beendocumented previously, including c-terminal Src kinase (CSK),CSK-homologous kinase (CHK), Wiscott-Aldrich syndrome protein (WASP),RACK1 and caveolin (20-23).

The Na/K-ATPase interacts directly with Src via at least two bindingmotifs: one being between the CD2 of the α1 subunit and Src SH2; and,other involving the third cytosolic domain (CD3) and Src kinase domain.The formation of this Na/K-ATPase and Src complex serves as a receptorfor ouabain to provoke protein kinase cascades. Specifically, binding ofouabain to Na/K-ATPase will disrupt the latter interaction, and thenresult in assembly and activation of different pathways including ERKcascades, PLC/PKC pathway and mitochondrial ROS production (24).Moreover, this interaction keeps Src in an inactive state. Thus, theNa/K-ATPase functions as an endogenous negative Src regulator. This isconsistent with the fact that the basal Src activity is inverselycorrelated to the amount of Na/K-ATPase α1 subunit in both culturedcells and in α1 heterozygous mouse tissues (25,26). See also, theco-inventors' pending application PCT/US07/023,011, filed Oct. 17, 2007(Pub. No. WO 2808/054792 on May 8, 2008), claiming priority from U.S.Ser. No. 60/855,482 filed Oct. 16, 2006, which are expresslyincorporated herein by reference.

There is still a need to determine the molecular interaction between theNa/K-ATPase and Src in order to then develop novel Src modulators thatmay be used to antagonize ouabain-induced signal transduction.

Moreover, there is a need for targeting the newly discoveredNa/K-ATPase/Src receptor complex to develop novel agonists orantagonists of the receptor so that the receptor function ofNa/K-ATPase/Src complex can be either stimulated for treating diseasessuch as ischemia/reperfusion injury or inhibited for treating diseasessuch as tissue fibrosis, congestive heart failure, and cancer.

Such a general method would be of tremendous utility in that wholefamilies of related proteins each with its own version of the functionaldomain of interest could be identified. Knowledge of such relatedproteins would contribute greatly to our understanding of variousphysiological processes, including cell growth or death, malignancy,renal/cardiovascular function and immune reactions.

Such a method would also contribute to the development of increasinglymore effective therapeutic, diagnostic, or prophylactic agents havingfewer side effects.

According to the present invention, just such novel compositions andmethods are provided.

SUMMARY OF THE INVENTION

In a broad aspect, there is provided herein a novel Src inhibitor,comprising a composition that targets the Na/K-ATPase/Src receptorcomplex and antagonizes ouabain-induced protein kinase cascades in oneor more cells in need thereof.

In a broad aspect, there is provided herein a novel Src inhibitor,comprising a composition that mimics the Na/K-ATPase-mediated regulationof Src and Src family kinases in one or more cells in need thereof.

In a broad aspect, there is provided herein a novel Src inhibitor,comprising a composition that inhibits Src in vesicular or cytosoliccompartments in one or more cells in need thereof.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIGS. 1A-1E: Identification of the N-terminus of N domain as aSrc-interacting motif from the Na/K-ATPase α1 subunit.

FIG. 1A: Schematic presentations of different GST-fusion proteins.

FIGS. 1B, 1D: The Coomassie blue staining of purified GST-ND, GST-CD3and GST-ND1, GST-ND1R, GST-ND2, GST-ND2R.

FIGS. 1C, 1E: Binding of GST-ND and GST-ND1 to Src. Purified His-Src(200 ng) was incubated with 5 μg GST fusion proteins in 0.5% TritonX-100 PBS for 30 min and followed by three washes with the same buffer.A representative Western blot from three independent experiments showedthe pulldown products probed with anti-His antibody.

FIGS. 2A-2D: Regulation of Src by ND1.

FIG. 2A: Soluble GST fusion proteins (100 ng) was incubated withrecombinant Src (4.5 U) for 15 min in PBS. Src activity was indicated bythe phosphorylation of Y418 in the presence of ATP/Mg²⁺.

FIG. 2B: Dose-dependent inhibition of Src by GST-ND1.

FIG. 2C-2D: Effects of YFP-ND1 and other fusion proteins on Src activityin LLC-PK1 cells. Cells were transiently transfected with pEYFP orpEYFP-ND1, pEYFP-ND, pEYFP-CD3 plasmids. 24 h after transfection,phosphorylation of Src in the cell lysates was measured by Western blotwith anti-pY418 antibody. Representative Western blots and combined datafrom 3 to 5 independent experiments were shown. **p<0.01 compared withthe control.

FIGS. 3A-3B: Targeting of YFP-ND1 to the Na/K-ATPase/Src complex in livecells.

FIG. 3A: Localization of YFP-ND1 in LLC-PK1 cells. LLC-PK1 cells weretransfected with pEYFP-ND1 and localization of YFP-ND1 was detected byconfocal microscope. Arrow indicated the plasma membrane localization ofYFP-ND1.

FIG. 3B: Lysates from transfected cells were immunoprecipitated withanti-Na/K-ATPase α1 antibody and then analyzed by Western blot usinganti-GFP antibody and anti-Na/K-ATPase α1 antibody. A representativeWestern blot of three separate experiments was shown.

FIGS. 4A-4F: Effect of ND1-derived peptides on Src activity.

FIG. 4A: Sequences of different ND1-derived peptides:

[SEQ ID NO: 1] peptide 1 . . . MTVAHMWFDNQIHEADTTEN; [SEQ ID NO: 2]peptide 2 . . . IHEADTTENQSGVSFDKTSA; [SEQ ID NO: 3]peptide 3 . . . SATWLALSRIAGLCNRAVFQ; [SEQ ID NO: 4]peptide 4 . . . ALSRIAGLCNRAVFQANQEN; [SEQ ID NO: 5]ND1 . . . LTQNRMTVAHMWFDNQIHEADTTENQSGVSFDKTSATW LALSRIAGLCNRAVFQANQEN.

FIG. 4B: Each peptide (1 μM) was incubated with recombinant Src (4.5 U)for 15 min, and then assayed for pY418 as in FIG. 1.

FIG. 4C: Dose-dependent inhibition of Src by the “NaKtide” (peptide 3).Curve fit analysis was performed with GraphPad software.

FIG. 4D: The effect of ATP concentration on the “NaKtide” (peptide3)-induced Src inhibition. The “NaKtide” (peptide 3) (0.1 μM) wasincubated with Src, and then assayed for pY418 in the presence ofdifferent concentrations of ATP/Mg²⁺.

FIG. 4E: Effects of the “NaKtide” (peptide 3) on Lyn kinase activity.Recombinant Lyn (100 ng) was incubated with indicated amount of peptide3 for 15 min, and then assayed for pY396 by Western blot. Curve fitanalysis was done with GraphPad software.

FIG. 4F: Effects of the “NaKtide” (peptide 3) on kinase activity of PKCmixture. Quantitative data were presented as mean±SE of at least threeindependent experiments. **p<0.01 compared with control.

FIGS. 5A-5E: Properties of cell permeable peptides.

FIG. 5A: Sequence information of different peptides:

[SEQ ID NO: 6] pC1 . . . GRKKRRQRRRPPQMTVAHMWFDNQIHEADTTEN;[SEQ ID NO: 7] pNaKtide . . . GRKKRRQRRRPPQSATWLALSRIAGLCNRAVFQ;[SEQ ID NO: 8] AP-NaKtide . . . RQIKIWFQNRRMKWKKSATWLALSRIAGLCNR AVFQ.[SEQ ID NO: 9] A1N-NaKtide . . . KKGKKGKKSATWLALSRIAGLCNRAVFQ

FIG. 5B: Dose-dependent inhibition of Src by the “pNaKtide” and control“pC1”. Curve fit analysis was performed by GraphPad software.

FIG. 5C, 5D: Cell loading analyses of the “pNaKtide” and “AP-NaKtide” inLLC-PK1 cells. Cells were serum-starved for 12 h and then exposed to 1μM of FITC-pNaKtide or FITC-AP-NaKtide at 37° C. for 60 min. Cells werewashed twice with PBS, and analyzed by confocal imaging. The scale barrepresents 20 μm.

FIG. 5E: Inhibition of Src by “A1N-NaKtide”.

FIGS. 6A-6C: Effects of the “pNaKtide” on the formation ofNa/K-ATPase/Src complex.

FIG. 6A: LLC-PK1 cells were transfected with EYFP-rat α1 (yellow) andSrc-ECFP (cyan). FRET analysis was performed as described herein. Regionof Interest 1 (Boxed area marked by ROI 1) was photobleached andanalyzed for FRET. The same measurement was done in ROI 2 that was notphotobleached.

FIGS. 6B, 6C: The same FRET analyses were conducted in transfected cellspretreated with 1 μM pC1 or different concentrations of “pNaKtide” for 1h. Average FRET efficiency (FIG. 6B) and percentage of cells (FIG. 6C)showing FRET efficiency (cut-off value is 4.0%) were calculated. Atleast 20 cells from three experiments were measured for each condition.

FIGS. 7A-7B: Effect of the “pNaKtide” on Src and Src-mediated signalingpathway.

FIG. 7A: Cells were serum-starved for 12 h and were exposed to 1 μM“pC1” or the “pNaKtide” for 1 h. Cell lysates were assayed by Westernblot. N=3. *p<0.05; **p<0.01 compared with control pC1.

FIG. 7B: SYF and SYF+Src cells were exposed to 1 μM “pC1” or the“pNaKtide”. Cell lysates were analyzed by Western blot. N=3. *p<0.05compared with control pC1.

FIGS. 8A-8C: Effect of the “pNaKtide” on ouabain-induced signaltransduction.

FIGS. 8A, 8B: LLC-PK1 (FIG. 8A) and primary cultured cardiac myocytes(FIG. 8B) were pre-incubated with 1 μM peptides for 1 h and then exposedto 100 nM (LLC-PK1) or 100 μM (myocytes) ouabain. Cell lysates wereanalyzed by Western blot. N=3. **p<0.01 compared with control peptide.

FIG. 8C: Primary cultured cardiac myocytes were pre-incubated with 1 μMpeptides for 1 h or PP2 for 30 min, and then exposed to 20 ng/ml IGF-1for 5 min. N=3. **p<0.01.

FIG. 9: Table 1 showing the effects of ND1, NaKtide, pNaKtide and PP2 onSrc.

FIG. 10A-10D: Correlation between Na/K-ATPase α1 amount and Srcactivity.

FIG. 10A: Expression level of Na/K-ATPase α1 in different cell lines.Various cells were harvested at 95% density and lysates were analyzed byWestern blot with anti-Na/K-ATPase α1 antibody.

FIG. 10B: Effects of Na/K-ATPase knockdown on Src activity in mouseliver. Liver samples from wild type (WT) and Na/K-ATPase α1 knockdown(+/−) mice were assessed by Western blot. N=6. **p<0.01.

FIG. 10C, 10D: Changes of Na/K-ATPase α1 amount in cells with differentdensities. Lysates of LLC-PK1 cells (FIG. 10C) and DU145 cells (FIG.10D) under indicated cell densities were analyzed with anti-Na/K-ATPaseα1, pY418 and Src antibodies.

FIG. 11A-11C: Regulation of Src and Src-mediated signaling by“pNaKtide”.

FIG. 11A: Regulation of basal FAK and ERK phosphorylation by “pNaKtide”.TCN23-19 cells were serum-starved for 12 h and then exposed to 1 μM“pNaKtide” for indicated times. FAK and ERK phosphorylation was assessedby Western blot with anti-pFAK576/7 and anti-phospho-ERK antibodies,respectively.

FIG. 11B: Effect of “pNaKtide” on Src in various cells. CulturedLLC-PK1, LNCaP, and DU145 cells were exposed to 1 μM pNaKtide for 1 h.Then, cells were fixed with cold methanol and immunostaining wasperformed with anti-Na/K-ATPase α1 and anti-pY418 antibodies.

FIG. 11C: Regulation of Src and Src-mediated signaling by “pNaKtide” inDU145 cells. Cell lysates were analyzed by Western blot with antibodiesagainst pY418, phosphor-ERK, pFAK576/7 and c-Myc. Membranes were stripedand reprobed with antibodies against Src, ERK, and FAK.

FIG. 12: Effect of “pNaKtide” on prostate cancer cell migration. Scrapedwound was introduced on confluent monolayer DU145 cells and the statusof wound closure in the absence or presence of “pNaKtide” was monitoredafter 24 h.

FIG. 13A, 13B: Effect of ND1 overexpression on cell viability in cancercells. DU145 cells (FIG. 13A) or MCF-7 cells (FIG. 13B) were transientlytransfected with plasmid constructs expressing YFP or YFP-ND1 usingLipofectAMINE 2000. Fluorescence and phase-contrast images werecollected at indicated time after transfection and then merged with SPOTVersion 4.6 software (Diagnostic Instruments). Small boxed regions wereenlarged and shown in the bottom right boxes. Original magnifications,×400. The same experiments were repeated at least three times.

FIG. 14A-14D: Effects of “pNaKtide” on cell viability in various cancercells. N=4. *p<0.05. **p<0.01.

FIG. 14A: “pNaKtide” caused dose-dependent inhibition of cell viabilityin LNCaP, DU145, and PC-3 prostate cancer cells.

FIG. 14B: Time-dependent effect of “pNaKtide” on DU145 cell viability.

FIG. 14C, 14D: Dose-dependent inhibition of cell viability inneuroblastoma cells (FIG. 14C) and breast cancer cells (FIG. 14D).

FIG. 15A-15D: Effects of “pNaKtide” on the growth of DU145 xenografttumors. 5×10⁶ DU145 prostate cells were injected subcutaneously in theflank of NOD/SCID mice. Tumor volume was estimated by calipermeasurements of the length (L) and width (W) as V=(L×W²)/2. After tumorvolume reaches 100 mm³, mice were treated by injecting different dosesof “pNaKtide” formulated in saline.

FIG. 15A: Effect of “pNaKtide” on tumorigenicity of DU145 cells inNOD/SCID mice. Tumors were removed and weighted after mice weresacrificed at 44 days. *, P<0.05. **, P<0.01.

FIG. 15B: Mice bearing xenograft tumors. Arrowheads identify thelocation of the tumors.

FIG. 15C: Growth of DU145 xenograft tumors in NOD/SCID mice treated withsaline or “pNaKtide”.

FIG. 15D: Inhibition of Src in “pNaKtide” treated xenograft tumors.After Xenograft tumors were removed and weighted, tumor homogenates wereassessed by Western blot. **, P<0.01.

FIG. 16A-16D: Effect of “pNaKtide” on angiogenesis. *, P<0.05. **,P<0.01.

FIG. 16A: Inhibition of endothelial cell proliferation by “pNaKtide”.Human Umbilical Vein Endothelial cells (HUVEC) and Human AorticEndothelial cells (HAEC) cultured on 12-well plates were exposed to“pNaKtide” with indicated concentrations for 72 h. Cell numbers werecounted with hemocytometer.

FIG. 16B: Immunohistostaining of CD31 in the formalin-fixed,paraffin-embedded xenograft tumors.

FIG. 16C: Effect on the tumor vessel density by “pNaKtide”. The vesseldensity was calculated as the percent of tumor area occupied by vessels.

FIG. 16D: Expression of VEGF in saline/pNaKtide-treated xenograftedtumor homogenates. Tumor homogenates were analyzed by Western blot withanti-VEGF antibody.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984). Unless otherwise defined, all terms of art, notationsand other scientific terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. As appropriate, procedures involving the use ofcommercially available kits and reagents are generally carried out inaccordance with manufacturer defined protocols and/or parameters unlessotherwise noted.

This invention is based, at least in part, on the inventors' discoverythat the Na/K-ATPase binds and inhibits Src. The inventors now revealthe molecular mechanism of Na/K-ATPase-mediated Src regulation.

In a broad aspect, the invention relates to the generation of a novelpeptide Src inhibitor from the Na/K-ATPase α1 subunit that targets tothe Na/K-ATPase/Src receptor complex and antagonizes ouabain-inducedprotein kinase cascades in cultured cells.

The invention is also based, at least in part, on the inventors'discovery that the Na/K-ATPase inhibits Src kinase by binding of theN-terminus of nucleotide binding domain to the Src kinase domain. Theinventors herein have now further discovered the identification of a 20amino acid peptide (NaKtide) that functions as an effective Srcinhibitor (IC₅₀=70 nM). Unlike small molecular Src inhibitors such asPP2, NaKtide is not an ATP analog, thus represents a novel class of Srcinhibitors. Moreover, it does not directly affect PKC and ERK familiesof serine/threonine kinases and it inhibits Lyn, another Src familytyrosine kinase, with a much lower potency (IC₅₀=2.5 μM).

The invention is also based, at least in part, on the inventors'discovery that highly positively charged leader peptide conjugatesincluding HIV-Tat-NaKtide (pNaKtide), Penetratin-NaKtide (AP-NaKtide),α1 N-terminus-NaKtide (A1N-NaKtide) readily enter cultured cells.

The invention is also based, at least in part, on the inventors'discovery, using the functional studies of pNaKtide, that this conjugatecan specifically target the Na/K-ATPase-interacting pool of Src and actsas a potent ouabain antagonist in cultured cells.

The invention is also based, at least in part, on the inventors'discovery, using the functional studies of AP-NaKtide, that thisconjugate can specifically target the intracellular vesicles and can actas a potent Src inhibitor and ouabain antagonist in cultured cells.

The invention is also based, at least in part, on the inventors'discovery, using the functional studies of NaKtide, that loading thecells with soluble NaKtide by either detergent or HIV-Tat-SS-NaKtide(ssNaKtide) conjugate can produce potent inhibition of cellular Srcactivity and acts as a potent ouabain antagonist in cultured cells.

The invention is also based, at least in part, on the inventors'discovery, using the functional studies of A1N-NaKtide, that thisconjugate can specifically target the intracellular compartments and canact as a potent Src inhibitor and ouabain antagonist in cultured cells.

pNaKtide, unlike PP2, resides mainly in the plasma membrane.Consistently, it affects much less the basal Src activity than that ofPP2. AP-NaKtide and A1N-NaKtide reside mainly in vesicles and also haveless effect on basal Src activity. On the other hand, ssNaKtide wouldhave significant effect on basal Src as PP2.

pNaKtide is effective in disrupting the formation of Na/K-ATPase/Srcreceptor complex in a dose-dependent manner. Consequently, it blocksouabain-induced activation of Src and a down-stream signaling pathwaysuch as ERK1/2 in cultured cells.

Unlike PP2, pNaKtide does not affect IGF-induced ERK activation incardiac myocytes. Thus, in another broad aspect, the present inventionrelates to the use of pNaKtide as a ouabain antagonist.

In another aspect, there are provided herein methods of using pNaKtidefor probing the physiological and pathological significance of theinventors' discovery of the signaling function of Na/K-ATPase and CTS.

The invention is also based, at least in part, on the inventors'discovery that some cancer cells express less Na/K-ATPase and havehigher Src activity.

The invention is also based, at least in part, on the inventors'discovery that pNaKtide and AP-NaKtide can mimic the Na/K-ATPase andinhibit Src and then FAK in cancer cells.

The invention is also based, at least in part, on the inventors'discovery that pNaKtide and AP-NaKtide are effective in inhibitingcancer cell migration.

The invention is also based, at least in part, on the inventors'discovery that pNaKtide can inhibit proliferation of endothelial cellsand prevent angiogenesis.

The invention is also based, at least in part, on the inventors'discovery that expression of Src inhibiting YFP-ND1 or addition ofeither pNaKtide or AP-NaKtide inhibits some cancer cell growth or causescell death.

The invention is also based, at least in part, on the inventors'discovery that pNaKtide is effective in blocking the growth ofxenografted prostate cancer in NOD/SCID mice.

In another broad aspect, there is provided herein a method forinhibiting Src activity or antagonizing CTS-induced signal transductionin a subject in need thereof, comprising administering an effectiveamount of a peptide derived from Na/K-ATPase or biologically activefragments thereof.

In another aspect, there is provided herein a composition that functionsas an effective Src inhibitor, is not an ATP analog, does not directlyaffect PKC and ERK families of serine/threonine kinases, and inhibitsLyn, a Src family tyrosine kinase, comprising one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof.

In another aspect, there are provided herein several highly positivelycharged leader peptide conjugates of NaKtide which readily enter cells.

In another aspect, there is provided herein a method to target NaKtideto different cellular compartments to achieve cellularcompartment-specific inhibition of Src, as exemplified by the followingconjugates: HIV-Tat-NaKtide (pNaKtide) to the plasma membrane;Penetratin-NaKtide (AP-NaKtide) and Na/K-ATPase α1-N-terminal-NaKtide(A1N-NaKtide) to vesicles; and HIV-Tat-S-S-NaKtide (ssNaKtide) to thecytosol.

In another aspect, there is provided herein a method for targeting theNa/K-ATPase-interacting pool of Src and acting as a potent ouabainantagonist in one or more cells in need thereof, comprisingadministering an effective amount of pNaKtide.

In another aspect, there is provided herein a method for targeting theSrc in vesicles and acting as a potent Src inhibitor or ouabainantagonist in one or more cells in need thereof, comprisingadministering an effective amount of AP-NaKtide or A1N-NaKtide.

In another aspect, there is provided herein a method for targeting Srcin whole cell and acting as a potent Src inhibitor or ouabain antagonistin one or more cells in need thereof, comprising administering aneffective amount of ssNaKtide.

In another aspect, there is provided herein a method for disrupting theformation of Na/K-ATPase/Src receptor complex in a dose-dependentmanner, comprising administering an effective amount of one or morepeptides derived from Na/K-ATPase or biologically active fragmentsthereof.

In another aspect, there is provided herein a method for blockingouabain-induced activation of Src and a down-stream signaling pathwaysuch as ERK1/2 in a subject in need thereof, comprising administering aneffective amount of one or more peptides derived from Na/K-ATPase orbiologically active fragments thereof.

In another aspect, there is provided herein use of one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof as aouabain antagonist.

In another aspect, there is provided herein a method for determining thephysiological and pathological significance of the inventors' discoveryof the signaling function of Na/K-ATPase and CTS, comprising using oneor more peptides derived from Na/K-ATPase or biologically activefragments thereof as a probe.

In another aspect, there is provided herein a Src inhibitor comprising acomposition capable of targeting the plasma membrane Na/K-ATPase/Srccomplex selected from one or more of SEQ ID NOs: 1, 2, 3, 4 and 5, orbiologically active fragments thereof.

In another aspect, there is provided herein a composition capable ofselectively targeting the Na/K-ATPase-interacting pool of Src and offunctioning as an effective ouabain antagonist in one or more cells inneed thereof, comprising one or more peptides derived from Na/K-ATPaseor biologically active fragments thereof.

In another aspect, there is provided herein a Src inhibitor that isspecific to Src, shows no direct effect on PKC family of kinases,comprising one or more of ND1, NaKtide, pNaKtide, AP-NaKtide,A1N-NaKtide, ssNaKtide and biologically active fragments thereof.

In another aspect, there is provided herein a specific ouabainantagonist, comprising one or more peptides derived from Na/K-ATPase orbiologically active fragments thereof tagged with a positively chargedleader peptide. In certain embodiments, the tagged peptide comprisesHIV-Tat or Penetratin or Na/K-ATPase α1 N-terminal peptide or otherpositively charged leader peptide tagged to NaKtide.

In another aspect, there is provided herein a method for determining thephysiology and/or probing the pathological significance of Na/K-ATPaseand endogenous CTS, comprising using one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof.

In another aspect, there is provided herein a new therapeuticcomposition for cardiovascular diseases where the Na/K-ATPase/Srcreceptor is over-stimulated, comprising one or more peptides derivedfrom Na/K-ATPase or biologically active fragments thereof.

In another aspect, there is provided herein a method of inducing cellgrowth inhibition in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of an inhibitor of Srcand a CTS antagonist.

In another aspect, there is provided herein a composition for preventingCTS-provoked signaling pathway, the composition comprising one or morepeptides derived from Na/K-ATPase or biologically active fragmentsthereof.

In another aspect, there is provided herein a method for substantiallyabolishing ouabain-provoked signaling transduction in the heart in asubject in need thereof, comprising administering an effective amount ofone or more peptides derived from Na/K-ATPase or biologically activefragments thereof.

In another aspect, there is provided herein use of one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof, inthe preparation of a medicament for the treatment of a cancer relateddisorder or a cardiac disease related disorder.

In another aspect, there is provided herein a pharmaceutical compositioncomprising one or more peptides derived from Na/K-ATPase or biologicallyactive fragments thereof, and a physiologically acceptable carrier.

In certain embodiments, the composition is adapted for use as atreatment for cardiac hypertrophy, tissue fibrosis and/or congestiveheart failure.

In certain embodiments, the composition is adapted for use as achemotherapeutic agent.

In certain embodiments, the composition is adapted for use as atreatment for a cancer related disorder.

In certain embodiments, the composition is adapted for use as atreatment for a cancer related disorder selected from one or more ofprostate cancer, breast cancer, and neuroblastoma.

In another aspect, there is provided herein a method of identifying acandidate compound for the treatment of a disorder associated with oneor more of cardiac hypertrophy, tissue fibrosis, congestive heartfailure or cancer, the method comprising: providing an assay fordetecting an interaction between Na/K-ATPase and Src which inhibits Srcactivity; conducting the assay with a test compound; and identifying atest compound that is a non ATP-competitive Src inhibitor and a CTSantagonist, wherein a test compound that significantly inhibits thedisorder is a candidate compound for the treatment of the disorder.

In another aspect, there is provided herein a method of identifying acandidate compound for the treatment of a disorder associated with oneor more of cardiac hypertrophy, tissue fibrosis, congestive heartfailure or cancer, the method comprising: providing a model of thedisorder; contacting the model with a test compound; detecting in thesample a level of: i) one or more peptides derived from Na/K-ATPase orbiologically active fragments and active Src thereof, ii) or Na/K-ATPaseor CTS binding; and comparing the level of the peptides and active Srcto a reference, wherein a test compound that causes a significantdifference in a level of the peptides as compared to the reference is acandidate compound for the treatment of the disorder.

In another aspect, there is provided herein a method of diagnosing asubject with a disorder associated with one or more of cardiachypertrophy, tissue fibrosis, congestive heart failure or cancer, themethod comprising: providing a sample from the subject; detecting in thesample a level of: i) one or more peptides derived from Na/K-ATPase orbiologically active fragments and active Src thereof, ii) or Na/K-ATPaseor CTS binding; and comparing the level of these parameters to areference, wherein a significant difference in a level of the peptidesas compared to the reference indicates that the subject has thedisorder.

In another aspect, there is provided herein a method of evaluating atreatment for a disorder associated with one or more of cardiachypertrophy, tissue fibrosis, congestive heart failure or cancer, themethod comprising: providing a sample from the subject; detecting in thesample a level of: i) one or more peptides derived from Na/K-ATPase orbiologically active fragments and active Src thereof, ii) or Na/K-ATPaseor CTS binding; and administering one or more doses of a treatment, andcomparing the level of the peptides and active Src to a reference,wherein a significant difference in a level of the peptides, as comparedto an unaffected individual, as compared to the reference indicates theefficacy of the treatment.

In certain embodiments, the reference represents a level of the peptidesprior to administration of the treatment.

In certain embodiments, the sample is from cardiac tissue or a cancercell of the subject.

In another aspect, there is provided herein a method of determining asubject's risk for development of a complication of a disorderassociated with one or more of cardiac hypertrophy, tissue fibrosis,congestive heart failure or cancer, the method comprising: providing asample from the subject; detecting in the sample a level of: i) one ormore peptides derived from Na/K-ATPase or biologically active fragmentsand active Src thereof, ii) or Na/K-ATPase or CTS binding; and comparingthe level of the peptides to a reference, wherein a significantdifference in a level of the peptides as compared to the referenceindicates the subject's risk of developing the complication.

In another aspect, there is provided herein a method of determining whena treatment for a disorder associated with one or more of cardiachypertrophy, tissue fibrosis, congestive heart failure or cancer, shouldbe initiated in a subject, the method comprising: providing a samplefrom the subject; detecting in the sample a level of: i) one or morepeptides derived from Na/K-ATPase or biologically active fragments andactive Src thereof, ii) or Na/K-ATPase or CTS binding; and comparing thelevel of to a reference, wherein a significant difference in a level ofthe peptides as compared to the reference indicates whether thetreatment should be initiated.

In certain embodiments, the reference represents a level of Na/K-ATPasepeptides or Na/K-ATPase or CTS binding in an unaffected subject.

In another aspect, there is provided herein a method for preventing ortreating a condition mediated by a ouabain steroid receptor in asubject, comprising administering one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof, and/or an agonistor antagonist thereof.

In certain embodiments, the condition is one or more of cancer, cardiachypertrophy, tissue fibrosis or congestive heart failure.

In another aspect, there is provided herein a method for identifying oneor more of: i) a substance that modulates a ouabain steroid receptor, aNa/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex, ii) aprocess mediated by a ouabain steroid receptor, a Na/K-ATPase receptorand/or a Na/K-ATPase/Src receptor complex, iii) degradation of a ouabainsteroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Srcreceptor complex, iv) a ouabain steroid receptor and/or Na/K-ATPasereceptor signaling transduction pathway, v) a condition mediated by aouabain steroid receptor, a Na/K-ATPase receptor and/or aNa/K-ATPase/Src receptor complex, vi) a steroid receptortransactivation, and/or inhibits or potentiates the interaction of aouabain steroid receptor, a Na/K-ATPase receptor and/or aNa/K-ATPase/Src receptor complex, comprising assaying for a substancethat inhibits or stimulates a ouabain steroid receptor, Na/K-ATPasereceptor and/or a Na/K-ATPase/Src receptor complex.

In another aspect, there is provided herein a method for evaluating asubstance, comprising: reacting one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof and a receptortherefore with a test substance, wherein the peptides and receptor bindto form a complex; and comparing to a control in the absence of the testsubstance to determine if the substance stimulates or inhibits thebinding of the peptides to the receptor.

A method of conducting a drug discovery business comprising: a)providing a method for identifying a substance identified using one ormore methods described herein; b) conducting therapeutic profiling ofsubstances identified in step a), or further analogs thereof, forefficacy and toxicity in animals; and c) formulating a pharmaceuticalpreparation including one or more substances identified in step b) ashaving an acceptable therapeutic profile.

In certain embodiments, the receptor is a ouabain receptor, aNa/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex.

In certain embodiments, a part of the peptide consists of a bindingdomain of the peptide that interacts with a ouabain receptor, whereinthe part is the N-terminus of nucleotide binding domain that binds theSrc kinase domain.

In another aspect, there is provided herein a method for regulating theNa/K-ATPase/Src receptor complex in a subject comprising inhibiting orstimulating the expression of one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof, a complex thereof;or the interactions thereof with a receptor.

In another aspect, there is provided herein a method for identifying oneor more conditions selected from: cardiac hypertrophy, tissue fibrosis,congestive heart failure or cancer, in a subject comprising detectingchanges in one or more peptides derived from Na/K-ATPase or Na/K-ATPase,CTS binding or biologically active fragments thereof in a sample fromthe subject.

In certain embodiments, the method comprising: collecting a sample fromthe subject; measuring the levels of one or more peptides in the sample;and comparing the levels of peptides in the sample to the levels insubjects not having cancer or a cardiac condition.

In certain embodiments, significantly decreased levels in the samplecompared to levels in samples from subjects who do not suffer from thecondition is indicative of an increased risk of the condition in thesubject.

In another aspect, there is provided herein agents, compounds, andsubstances identified using the methods described herein. The agents,compounds, and substances are useful in the treatment or prevention of acondition mediated by a steroid receptor, including a condition mediatedby a ouabain receptor.

In another aspect, there is provided herein antibodies specific forpeptides derived from Na/K-ATPase or biologically active fragmentsthereof.

In another aspect, there is provided herein antibodies labeled with adetectable substance and used to detect proteins or complexes derivedfrom Na/K-ATPase or biologically active fragments thereof in biologicalsamples, tissues, and cells.

In another aspect, there is provided herein antibodies having uses intherapeutic applications, and in conjugates and immunotoxins as targetselective carriers of various agents which have therapeutic effectsincluding chemotherapeutic drugs, toxins, immunological responsemodifiers, enzymes, and radioisotopes.

In another aspect, there is provided herein a pharmaceutical compositionadapted for administration to a subject for the prevention or treatmentof a condition mediated by a steroid receptor comprising an effectiveamount of one or more peptides derived from Na/K-ATPase or biologicallyactive fragments thereof, or agonists or antagonists thereof, or anagent, compound or substance identified using a method described herein,and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, there is provided herein a pharmaceutical compositionadapted for the treatment of a patient suffering from a cardiac orcancer disorder which comprises a therapeutically effective amount ofone or more peptides derived from Na/K-ATPase or biologically activefragments thereof, or a pharmaceutically acceptable salt thereof.

In another aspect, there is provided herein a pharmaceutical compositioncomprising an effective amount of one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof or an agonist orantagonist thereof, and an appropriate carrier, diluent, or excipient.

In another aspect, there is provided herein a pharmaceutical compositionadapted for administration to a subject for the prevention or treatmentof a condition mediated by a steroid receptor, in particular a conditionmediated by a ouabain receptor, and an appropriate carrier, diluent, orexcipient.

In another aspect, there is provided herein use of peptides derived fromNa/K-ATPase, biologically active fragments thereof or agonists orantagonists thereof, for the manufacture of, or in the preparation of amedicament.

In another aspect, there is provided herein a substance for inhibitingouabain-provoked signal transduction comprising one or more of thepeptides, or a complex thereof. In certain embodiments, the complex issubstantially cell permeable.

In non-limiting examples, the present invention relates to a compositionof matter comprising an amino acid peptide comprising at least tenconsecutive amino acid residues of the sequence SATWLALSRIAGLCNRAVFQ[SEQ ID NO: 3], or conservative substitutions of one or more amino acidresidues, or substitutions with unnatural amino acids to improvepharmacodynamics or/and pharmacokinetics, wherein the peptide is capableof binding the kinase domain of Src.

Included within the scope of the invention are compositions whichfurther comprise a therapeutically acceptable excipient. In certainembodiments, the amino acid peptide comprises the sequenceSATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3].

In other embodiments, the amino acid peptide comprises a sequenceselected from the group consisting of: SEQ ID NO: 3; SEQ ID NO: 4; andSEQ ID NO: 5.

In particular embodiments, the amino acid peptide comprises SEQ ID NO 7.In other particular embodiments, the amino acid peptide comprises SEQ IDNO: 8 or SEQ ID NO: 9.

Included within the scope of the invention are nucleic acid sequencesencoding a composition as described herein.

Included within the scope of the invention are vectors comprising anucleic acid sequence as described herein. In certain embodiments, thevector comprises pEYFP.

Included within the scope of the invention are cells comprising a vectoras described herein. In certain embodiments, the cell is E. coli. Incertain embodiments, the cell is mammalian. In certain embodiments, thecell is a tumor cell.

In another aspect, there is provided herein a monoclonal antibodyselective for a composition as described herein. In certain embodiments,the monoclonal antibody further comprises a detectable label selectedfrom the group consisting of: radioactive label; chemical label;fluorescent label; an antibody; and a protein.

Included within the scope of the invention are compositions where thecomposition is capable of affecting a cellular process selected from thegroup consisting of: antagonizing a CTS-induced protein kinase cascade;upregulating a CTS induced protein kinase cascade; Src inhibition; Srcstimulation; Na/K-ATPase mimic; Na/K-ATPase competitive inhibitor; Lyninhibition; Lyn stimulation; ouabain antagonism; ouabain stimulation;ERK1/2 activation; ERK1/2 inhibition; membrane permeability by sodiumions; membrane permeability by potassium ions. In certain embodiments,the composition is not an ATP analog.

Included within the scope of the invention are compositions whichfurther comprise means to therapeutically permeate plasma membrane.

Included within the scope of the invention are compositions furthercomprise at least one additional therapeutic composition useful to atreat a disease selected from the group consisting of: cancer; vasculardisease; cardiovascular disease; heart disease; prostate cancer; breastcancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis; congestiveheart failure; ischemia/reperfusion injury.

In certain embodiments, the composition further comprises a secondcompound bound with the amino acid peptide in a location other than SEQID NO: 3, wherein the second compound is selected from the groupconsisting of: chemotherapeutic drug; toxin; immunological responsemodifier; enzyme; and radioisotope.

In certain embodiments, the composition further comprises a secondcompound bound with the amino acid peptide in a location other than SEQID NO: 3, wherein the second compound is selected from the groupconsisting of: HIV-Tat; Penetratin; and HIV-Tat-S-S; GST.

In certain embodiments, the composition comprises HIV-Tat-SEQ ID NO: 3.

In certain embodiments, the composition comprises a fusion protein,provided that the fusion does not disrupt the at least ten consecutiveresidues of SEQ ID NO: 3. In certain embodiments, the fusion is withGST.

In another aspect, there is provided herein a method to bind a compoundto the kinase domain of Src in a Src-expressing cell, comprisingcontacting a compound described herein to at least one Src-expressingcell. In certain embodiments, the Src-expressing cell is a mammaliancell.

In certain embodiments, the at least one mammalian cell is a cellselected from the group consisting of: heart cell, liver cell, vascularcell; breast cell; prostate cell; kidney cell; muscle cell; blood cell;and brain cell. In certain embodiments, the at least one mammalian cellis cultured in vitro. In certain embodiments, the at least one mammaliancell is an animal model. In certain embodiments, the at least onemammalian cell is a human.

In another aspect, there is provided herein a method of treating aSrc-associated disease in a mammal in need of such treatment, comprisingadministering a therapeutic composition described herein. In certainembodiments, the Src-associated disease is selected from the groupconsisting of: cancer; vascular disease; cardiovascular disease; heartdisease; prostate cancer; breast cancer; neuroblastoma; cardiachypertrophy; tissue fibrosis; congestive heart failure; andischemia/reperfusion injury. In certain embodiments, the mammal is ahuman. In certain embodiments, the therapeutic composition comprises anamino acid peptide comprising a sequence selected from the groupconsisting of: SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 7;SEQ ID NO: 8; and SEQ ID NO: 9.

In another aspect, there is provided herein a method of treating cancerin a mammal in need of such treatment, comprising administering aSrc-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treatingvascular disease in a mammal in need of such treatment, comprisingadministering a Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treatingcardiovascular disease in a mammal in need of such treatment, comprisingadministering a Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treating heartdisease in a mammal in need of such treatment, comprising administeringa Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treatingprostate cancer in a mammal in need of such treatment, comprisingadministering a Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treating breastcancer in a mammal in need of such treatment, comprising administering aSrc-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treatingneuroblastoma in a mammal in need of such treatment, comprisingadministering a Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treating cardiachypertrophy in a mammal in need of such treatment, comprisingadministering a Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treating tissuefibrosis in a mammal in need of such treatment, comprising administeringa Src-inhibiting therapeutic composition described herein.

In another aspect, there is provided herein a method of treatingcongestive heart failure in a mammal in need of such treatment,comprising administering a Src-stimulating therapeutic compositiondescribed herein.

In another aspect, there is provided herein a method of treatingischemia/reperfusion injury in a mammal in need of such treatment,comprising administering a Src-stimulating therapeutic compositiondescribed herein.

In another aspect, there is provided herein a method to reduce increasedbasal Src activity in a tumor cell, comprising administering aSrc-inhibiting composition described herein to a Src-expressing tumorcell.

In another aspect, there is provided herein a method to inhibit FAK in atumor cell comprising administering a Src-inhibiting compositiondescribed herein to a Src-expressing tumor cell. In certain embodiments,the Src-expressing cell is a TCN23-19 cell.

In another aspect, there is provided herein a method to reduce tumorcell migration in a tumor cell test model, comprising administering aSrc-inhibiting composition described herein to a Src-expressing tumorcell.

In another aspect, there is provided herein a method to kill cancercells when the expression of Na/K-ATPase is reduced, comprisingadministering a Src-inhibiting composition described herein to aSrc-expressing tumor cell having reduced Na/K-ATPase expression.

In another aspect, there is provided herein a method of inhibiting cellgrowth in a tumor cell line, comprising administering a Src-inhibitingcomposition described herein to a Src-expressing tumor cell line.

In another aspect, there is provided herein a method which furthercomprises comparison of the ability of a composition described herein toinhibit cell growth in a tumor cell line to a test compound's ability toinhibit cell growth in the same tumor cell line.

In another aspect, there is provided herein a method of inhibitingprostate tumor cell growth in a prostate tumor cell line, comprisingadministering a Src-inhibiting composition described herein to aSrc-expressing prostate tumor cell line.

In another aspect, there is provided herein a method which furthercomprises comparison of the ability of a composition described hereininhibiting prostate tumor cell growth in a prostate tumor cell line to atest compound's ability to inhibit cell growth in the same prostatetumor cell line.

In another aspect, there is provided herein a method of inhibitingbreast tumor cell growth in a breast tumor cell line, comprisingadministering a Src-inhibiting composition described herein to aSrc-expressing breast tumor cell line.

In certain embodiments, the method further comprises comparison of theability of a composition described herein inhibiting prostate tumor cellgrowth in a breast tumor cell line to a test compound's ability toinhibit cell growth in the same breast tumor cell line.

In another aspect, there is provided herein a method of inhibitingneuroblastoma cell growth in a neuroblastoma tumor cell line, comprisingadministering a Src-inhibiting composition described herein to aSrc-expressing neuroblastoma tumor cell line.

In certain embodiments, the method further comprises comparison of theability of a composition described herein inhibiting neuroblastoma tumorcell growth in a prostate tumor cell line to a test compound's abilityto inhibit cell growth in the same neuroblastoma tumor cell line.

In another aspect, there is provided herein a method for screening atleast one test composition to determine whether the at least onecomposition affects Src, comprising: introducing a test compositioncomprising a modified amino acid peptide of SATWLALSRIAGLCNRAVFQ [SEQ IDNO: 3] to Src, wherein the modification is at least one conservativeamino acid substitution; and determining whether the test compositionaffects Src.

In certain embodiments, the affect is selected from the group consistingof: Src binding; Src inhibition; Src stimulation; Src function; Lynbinding; Lyn function; Lyn inhibition; ouabain antagonism; Na/K-ATPasefunction; ERK1/2 function; FAK inhibition.

In certain embodiments, the method includes introducing a testcomposition is accomplished in vitro.

In certain embodiments, the method includes introducing a testcomposition is accomplished in at least one mammalian cell.

In certain embodiments, the method includes introducing a testcomposition is accomplished in at least one tumor cell line.

In certain embodiments, the method includes determining whether thecomposition affects Src is measured by cell growth compared to control.

In certain embodiments, the method includes determining whether thecomposition affects Src is measured by cell migration compared tocontrol.

In certain embodiments, the at least one mammalian cell is an animalmodel. In certain embodiments, the animal model is a NOD/SCID mouse. Incertain embodiments, determining whether the composition affects Src ismeasured by tumor growth compared to control. In certain embodiments,the at least one mammalian cell is a human.

In certain embodiments, the method includes determining whether thecomposition affects Src is measured by tumor growth compared to control.

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. All publications, including patentsand non-patent literature, referred to in this specification areexpressly incorporated by reference. The following examples are intendedto illustrate certain preferred embodiments of the invention and shouldnot be interpreted to limit the scope of the invention as defined in theclaims, unless so specified.

-   -   Example I

Materials. Chemicals of the highest purity and culture media werepurchased from Sigma (St. Louis, Mo.). PP2, a Src kinase inhibitor, andstaurosporine, a non-specific PKC inhibitor, were obtained fromCalbiochem (San Diego, Calif.). The following antibodies were obtainedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.): monoclonal anti-Srcantibody (B12), polyclonal anti-ERK antibody, monoclonalanti-phosphor-ERK antibody, polyclonal anti-CD31 antibody, goatanti-rabbit and goat anti-mouse secondary antibodies. The monoclonalanti-His antibody was from GE Healthcare (Buckinghamshire, England).Polyclonal antibodies against phosphor-Akt (Ser473), Phosphor-FAK(576/7), Akt and FAK were purchased from Cell Signaling Technology(Danvers, Mass.). The polyclonal anti-VEGF antibody and monoclonalanti-α1 antibody (α6F) was obtained from Abcam (Cambridge, Mass.) andthe Developmental Studies Hybridoma Bank at the University of Iowa (IowaCity, Iowa), respectively. For purification of GST-fused proteins andHis-tagged proteins, Glutathione beads from Amersham Bioscience(Uppsala, Sweden) and ProBond Purification System from Invitrogen(Carlsbad, Calif.) were used respectively. Recombinant human Src and Lynexpressed in Sf9 insect cells for kinase activity assay and IGF-1expressed in Escherichia coli were obtained from Upstate Biotechnology(Lake Placid, N.Y.). Plasmids pEYFP-C1 was purchased from Clontech (PaloAlto, Calif.), and pGEX-4T-1 and pTrc-His A vectors were from Invitrogen(Carlsbad, Calif.). The Optitran nitrocellulose membranes used forWestern blotting were obtained from Schleicher and Schuell (Dassel,Germany). All the peptides were synthesized with the purity of 95%.Identity and purity were confirmed by high-performance liquidchromatography-mass spectroscopy.

Plasmid Constructs. The preparation of plasmid constructs expressing GSTfusion proteins were done as described (24). GST-CD3 (amino acid residue350-785), GST-ND (amino acid residue 379-594), GST-ND2 (amino acidresidue 379-475), GST-ND2R (amino acid residue 476-594), GST-ND1 (aminoacid residue 379-435) and GST-ND1R (amino acid residue 436-594)expression vectors were constructed based on the sequence of pig kidneyNa/K-ATPase α1 subunit. His-tagged Src constructs were generated byexcising the corresponding Src cDNA from the GST-Src vector (27) andthen inserting them into pTrc-His A vector. pEYFP-ND1, pEYFP-ND andpEYFP-CD3 were made by directional subcloning the corresponding cDNAsfrom the GST-Src vector into pEYFP-C1 vector. All constructs wereverified by DNA sequencing.

Cell Preparation, Culture and Transient Transfections. Pig kidneyproximal LLC-PK1, and mouse fibroblast SYF and SYF+Src cells wereobtained from American Type Culture Collection (Manassas, Va.) andcultured in DMEM medium containing 10% fetal bovine serum and penicillin(100 U/ml)/streptomycin (100 μg/ml). Na/K-ATPase α1 knockdown cellsPY-17 and TCN23-19 were generated from LLC-PK1 cells as described (25).LLC-PK1, PY-17 and TCN23-19 cells were serum-starved for 24 h, whereasSYF and SYF+Src cells were cultured in the medium containing 0.5% FBSfor 24 h and used for the experiments. All neuroblastoma cells, breastcancer cells, colon cancer cells, and prostate cancer cells wereobtained from American Type Culture Collection and maintained accordingto the instructions. Transient transfections were performed usingLipofectAMINE 2000 (Invitrogen) according to the manufacturer'sinstructions. Experiments were performed 24 h after transfection.

Primary cultures of neonatal rat cardiac myocytes were prepared asdescribed previously with minor modifications (28). Myocytes weredispersed from ventricles of 1- to 2-day-old Sprague-Dawley rats bydigestion with 0.04% collagenase II (Worthington) and 0.05% pancreatin(Sigma) at 37° C. Noncardiomyocytes were eliminated by preplating for1.5 h at 37° C. Myocytes were plated at a density of 8×10² cells/mm² in100-mm Corning cell culture dishes in Dulbecco's modified Eagle'smedium-M199 (4:1) containing 10% (vol/vol) fetal bovine serum (24 h, 37°C.) and then incubated in serum-free medium for 48 h beforeexperimentation. All research on rats was done according to proceduresand guidelines approved by the Institutional Animal Care and UseCommittee.

Preparation of Src, Na/K-ATPase, GST-fused Proteins, and His-taggedProteins. Src, without the first 85 amino acid residues, was purifiedfrom sf-9 cells as described (27) and used in the initial binding assaysto ensure that Src binds to the Na/K-ATPase. In the Src phosphorylationexperiment, purified recombinant full-length Src from UpstateBiotechnology was used. Na/K-ATPase was purified from pig kidney outermedulla using the Jorgensen method as we previously described (27) andthe preparations with specific activities between 1200 and 1400 μmmolPi/mg/h were used. GST-fused proteins or His-tagged proteins wereexpressed in Escherichia coli BL21 (Invitrogen) and purified usingglutathione beads or ProBond Purification System (Invitrogen). SolubleGST-fused proteins were eluted from the glutathione beads with elutionbuffer [10 mM reduced glutathione, 0.1% Triton X-100, 50 mM Tris-HCl,(pH8.0)] and then dialyzed in the buffer containing 0.1% Triton X-100,50 mM Tris-HCl (pH8.0) to remove remnant glutathione.

In vitro GST Pulldown Assay. GST pulldown assay were performed asfollowing: 5 μg GST-fused proteins were conjugated on glutathione beadsand incubated with 100 ng purified his-Src in 500 μl PBS in the presenceof 0.5% Triton X-100 at room temperature for 30 min. The beads werewashed with the same buffer for four times. The bound his-Src wasresolved on 10% SDS-PAGE and detected by Western blot with anti-Hisantibody.

Kinase Activity Assay of Src and Lyn. To determine how Na/K-ATPaseconstructs or peptides affect Src/Lyn kinase activity, the purified Src(4.5 U) or Lyn (20 ng) was incubated with different amount of thepurified GST-fused Na/K-ATPase constructs or peptides in PBS for 30 minat 37° C. Afterward, 2 mM ATP/Mg²⁺ was added. The reaction continued for5 min at 37° C. and was stopped by addition of SDS sample buffer.Afterward, the Src pY418 and Lyn pY396 were measured by anti-pY418antibody to indicate Src/Lyn activation (27). For the Src activity assayin TCN23-19 cells and primary rat neonatal cardiac myocytes, cells werelysed in ice-cold RIPA buffer containing 1% Nonidet P40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 μg/ml aprotinin, 10μg/ml leupeptin, and 50 mM Tris-HCl (pH 7.4). Cell lysates were clearedby centrifugation at 16,000×g for 15 min, and the supernatants wereseparated by SDS-PAGE (60 μg/lane) and transferred to an Optitranmembrane and were analyzed with anti-pY418 antibody. The pY418 signalwas detected using the enhanced chemiluminescence kit (Pierce) andquantified using a Bio-Rad GS-670 imaging densitometer as we previouslydescribed (29).

PKC Kinase Activity Assay. Activity of PKC was measured by PepTagphosphorylation assay for non-radioactive detection of PKC (Promega) asdescribed in the product instruction. Briefly, 40 ng PKC were incubatedfor 30 min at 30° C. with the reaction mixture containing 5 μl reactionbuffer, 5 μl PepTag C1 (0.4 μg/μl), 5 μl PKC activator solution, 1 μlpeptide protection solution and 10 μM staurosporine or peptide. Then thereaction mixture was subjected to electrophoresis on a 0.8% agarose gelat 100 V for 20 min. After electrophoresis, negatively-chargedphosphorylated PepTagC1 peptide migrated toward the anode (+), whilenon-phosphorylated PepTagC1 peptide migrated toward cathode (−).Percentage of the phosphorylated PepTagC1 was an indicator of the PKCactivity.

Immunoblot Analysis. Following the indicated treatment, the incubationmedium was rapidly replaced with ice-cold PBS. The washed cells werethen lysed in ice-cold RIPA buffer, and subjected to Western blotanalysis with anti phosphor-Akt (Ser473), phosphor-MAPK and phosphor-FAK(Tyr576/577) antibodies as described above. Then the same membrane wasstripped and reprobed with anti Akt, MAPK and FAK antibodiesrespectively.

Image Analysis with Confocal Fluorescence Microscope. Cells cultured oncoverslips were subjected to the indicated treatment. Then cells werewashed twice with PBS and fixed for 15 min with methanol prechilled at−20° C. The fixed cells were then rinsed with PBS three times andblocked with 200 μl of Image-iT FX signal enhancer (Invitrogen) for 30min at room temperature. The cells were washed again and incubated withanti-pY418 antibody in PBS containing 1% bovine serum albumin for 1 h atroom temperature. After three washes with PBS, the cells were incubatedwith Alexa Fluor-conjugated anti-rabbit secondary antibody (Invitrogen).Image visualization was performed using a Leica DMIRE2 confocalmicroscope (Leica, Mannheim, Germany). Leica confocal software was usedfor data analysis.

Localization Analysis of pNaKtide in Live Cells. Cells were cultured oncoverslips and then subjected to the indicated treatment ofFITC-pNaKtide at 37° C. Cells were washed twice with PBS andlocalization of pNaKtide was assessed by directly monitoring FITCfluorescence with Olympus fluorescence microscopy (Olympus).

Cell Viability Assay. After confluence reaches about 60%, cells wereexposed to peptides with indicated concentration and time. Cells werethen trypsinized and the cell suspensions were mixed with trypan blue atroom temperature for 5 m. The numbers of viable cells were counted withhemocytometer.

Wound Closure Assay. DU145 cells were seeded in 6-well plates in growthmedium containing 10% fetal bovine serum. The cells were allowed to growto confluent monolayer. The wound-induced migration was triggered byscraping the cells with a plastic pipette tip. The cells were then weretreated with or without pNaKtide at different concentrations. The woundwas imaged immediately (0 h) and at 24 h with an inverted phase-contrastmicroscope with a ×10 objective.

Establishment of DU-145 Xenograft Tumors in NOD/SCID Mice. NOD/SCID micenude mice (NCI) were housed in laminar airflow cabinets underpathogen-free conditions with a 12 h light/12 h dark schedule and fedautoclaved standard chow and water. Xenografts of DU145 were initiatedby subcutaneous injection of 5×10⁶ DU145 cells into the left and rightflanks of female nude mice at age of 4-6 weeks. Tumor volume wasestimated by caliper measurements of the length (L) and width (W) asV=(L×W²)/2. Treatment was started after tumors reached an average volumeof 100 mm³. Mice were injected subcutaneously near the tumor site withpNaKtide in saline at the dose of 2 mg/kg and 10 mg/kg (body weight)every other day for one week. All research on NOD/SCID mice was doneaccording to procedures and guidelines approved by the InstitutionalAnimal Care and Use Committee of University of Toledo Health ScienceCampus.

Analysis of Data. Data are given as the mean±SE. Statistical analysiswas performed using the Student's t test, and significance was acceptedat p<0.05.

Results

Identification of ND1 as a Src-interacting domain from the Na/K-ATPasea1 subunit: The CD3 interacted and inhibited Src (24). As depicted inFIG. 1A, the Na/K-ATPase CD3 consists of both P and N domains. The 3Dstructure of Na/K-ATPase indicates that the N domain is exposed, whereasthe P domain is relatively close to the membrane (3,5,6). Thus, whilenot wishing to be bound by theory, inventors herein now believe that theN domain interacts with the Src kinase domain. To test, the inventorsconstructed GST-ND and tested whether it binds the purified His-Src.GST-CD3 was used as a positive control whereas purified GST served as anegative control. Pull-down analyses confirmed that the N domain of α1subunit interacted with Src (FIG. 1C).

As depicted in FIG. 1A, the N domain contains over 200 amino acidresidues. To further map the binding motif in the N domain, theinventors constructed ND2 and ND2 remaining (ND2R) GST fusion proteinsas illustrated in FIG. 1A. GST pull-down assay showed that GST-ND2, butnot GST-ND2R, bound Src (FIG. 1E).

Further structural analysis of the ND2 reveals that the N-terminus ofND2 is highly unstructural and may undergo induced-fit (30). Moreover,this domain is also highly exposed and is less important for ATP binding(31), and thus the catalytic function of the Na/K-ATPase. Thus, to testwhether the N-terminus of ND interacts with Src, the inventorsconstructed two more GST-fusion proteins (ND1 and ND1R), and assessedtheir binding to Src using GST pull-down assay. As depicted in FIG. 1E,the ND1, but not the ND1R, interacted with Src.

ND1 as a potent Src inhibitor can target to the Na/K-ATPase/Src complexin cultured cells: To test whether ND1 inhibits Src as did the CD3, theinventors incubated Src with 100 ng soluble GST fusion proteins in thetest tube and measured Src pY418 level by Western blot. GST-CD3 was usedin the experiment as a positive control and GST-ND was also tested.

As depicted in FIG. 2A, both GST-ND1 and GST-ND were as effective as thepositive control in inhibiting Src activity. Moreover, the inhibitoryeffect of ND1 on Src was dose-dependent (IC₅₀=50 nM) (FIG. 2B). Tofurther test whether ND1 can be used as a minigene product to inhibitSrc in live cells, the inventors measured Src pY418 level changes inLLC-PK1 cells transfected with different YFP expression vectors. Theexpression of YFP-ND1, but not YFP, reduced Src activity in cell lysatesas did by YFP-ND and YFP-CD3 (FIG. 2C, 2D).

These examples demonstrate the effectiveness of ND1 as a Src inhibitorin cultured cells. To further test whether ND1 can target the plasmamembrane Na/K-ATPase/Src complex, the inventors performed the followingthree sets of experiments.

First, as depicted in FIG. 3A, the YFP-ND1 was expressed as a solubleprotein. However, the inventors did detect a pool of YFP-ND1 residednear the plasma membrane. Second, to test whether this pool of YFP-ND1interacts with Src, the inventors co-transfected LLC-PK1 cells withYFP-ND1 and Src-CFP, and then performed FRET analysis. A significantFRET was detected in the cotransfected cells (13.4±2.4%), which showedthat YFP-ND1 and the plasma membrane Src-CFP were likely to associate.Finally, when cell lysates were immunoprecipitated by an anti-α1antibody, the inventors found that YFP-ND1 was co-precipitated (FIG.3B). Thus, these examples show that YFP-ND1 is most likely capable ofinteracting with the Na/K-ATPase-associated Src.

Development of NaKtide as a specific Src inhibitor: Since the above datademonstrated that ND1 binds and inhibits Src, the inventors synthesizedfour 20 mer peptides that cover the entire ND1 and tested which one actslike ND1, capable of inhibiting Src (FIG. 4A).

Based on the crystal structure of Na/K-ATPase (6), peptide 1 and 2 areun-structural whereas peptides 3 and 4 may form α-helix. As depicted inFIG. 4B, 1 μM peptide 3 caused almost a 100% inhibition of Src whilepeptide 4 produced a partial inhibition. On the other hand, both peptide1 and peptide 2 showed no effect. When the dose-response curve wasconstructed, the inventors showed that peptide 3 was quite potent ininhibiting Src with an IC₅₀ of about 70 nM (FIG. 4C), comparable to thatof GST-ND1. The peptide 1 had no effect. Since peptide 3 is derived fromthe Na/K-ATPase, the inventors named it “NaKtide”. Because peptide 1 hasno effect on Src, it was used as a control. Thus, the inventors named it“C1.”

To show that NaKtide acts as an ATP analog as a generic Src inhibitorPP2, the inventors measured its effect on Src in the presence ofdifferent concentrations of ATP. As shown in FIG. 4D, changes in ATPconcentration from 0.1 to 2 mM did not affect NaKtide-induced Srcinhibition.

To probe whether NaKtide is relatively specific to Src, the inventorsmeasured its dose-dependent effect on another Src family kinase Lyn. Asshown in FIG. 4E, the NaKtide produced a dose-dependent inhibition ofLyn. However, the IC₅₀ is about 2.5 μM, 40 times higher than that of Srcinhibition.

To test whether NaKtide affects serine/threonine kinases, the inventorsincubated a PKC family kinase cocktail with NaKtide and C1, and thenmeasured for the kinase activity. As shown in FIG. 4F, unlikestaurosporine (a non-specific PKC family kinase inhibitor), NaKtide, aswell as C1, showed no effect up to 10 μM.

Development of a cell-permeable NaKtide as a ouabain antagonist: Recentstudies have demonstrated that the coupling of biological molecules tovarieties of positively charged cell-penetrating peptides can facilitatetheir uptake into cultured mammalian cells as well as animal tissues(32,33). Accordingly, the inventors synthesized HIV-Tat-NaKtide(“pNaKtide”) (see FIG. 5A).

HIV-Tat-P1 (pC1) was also synthesized and used as a control. Meanwhile,after careful review on the Na/K-ATPase α1 sequence, the inventors foundan N-terminal polybasic fragment which may facilitate the uptake ofNaKtide. The inventors synthesized the Na/K-ATPase α1 N-terminal-NaKtide(A1N-NaKtide). To compare, the inventors also synthesizedPenetratin-NaKtide (“AP-NaKtide”). In vitro kinase assay showed thatNaKtide was a highly potent Src inhibitor while the control pC1 wasinactive (FIG. 5B). Interestingly, addition of HIV-Tat actuallyincreased the potency of the “NaKtide” (peptide 3). The IC₅₀ wasdecreased from 70 nM to about 5 nM. This was also true for AP-NaKtide(data not shown) and A1N-NaKtide (FIG. 5E).

To assess the cell permeability, both pNaKtide and AP-NaKtide werelabeled with FITC. As depicted in FIG. 5C, confocal imaging analysis oflive cells indicated that pNaKtide, but not NaKtide, was cell-permeable.Maximal loading was achieved after 30 to 60 min of incubation with 1 μMpNaKtide in almost every LLC-PK1 cell in culture. Moreover, unlikeYFP-ND1, pNaKtide resided mainly in the plasma membrane with somedistribution to the intracellular membrane compartments. On the otherhand, when the same experiments were conducted to assess AP-NaKtide,most of AP-NaKtide was in intracellular vesicles. Unlike pNaKtide, veryfew of AP-NaKtide was detected in the plasma membrane (FIG. 5D). Thus,different tags are likely to direct different intracellular distributionof NaKtide.

To test the effectiveness of pNaKtide as a potential ouabain antagonist,the inventors performed FRET analysis to determine the effect of NaKtideon the formation of Na/K-ATPase/Src receptor complex. LLC-PK1 cells wereco-transfected with YFP-α1 and Src-CFP, and then exposed to differentconcentrations of pNaKtide. The inventors focused on pNaKtide becausethis conjugate resided mainly in the plasma membrane where the receptorNa/K-ATPase/Src is located.

As depicted in FIG. 6A, both YFP-α1 and Src-CFP were targeted to theplasma membrane. Moreover, a significant FRET was detected in controlLLC-PK1 cells. Addition of pNaKtide produced a dose-dependent reductionin overall FRET efficiency (FIG. 6B) as well as the percentage of cellsthat exhibited detectable FRET (FIG. 6C), indicating that pNaKtide iseffective as YFP-ND1 in interacting with the plasma membrane pool ofSrc, thus blocking the formation of a stable Na/K-ATPase/Src complex.Since significant effect was observed when the cells were exposed to 100nM to 1 μM pNaKtide, 1 μM pNaKtide was used in the following three setsof experiments to test its effectiveness and specificity as a ouabainantagonist.

First, to assess how pNaKtide affects basal Src and ERK1/2 activity, theinventors exposed LLC-PK1 and the Na/K-ATPase-knockdown TCN23-19 cellsto 1 μM pC1 or pNaKtide for different times. Cell lysates were thensubjected to Western analysis of active Src and ERK1/2. TCN23-19 cellswere derived from LLC-PK1 cells (25). Knockdown of the Na/K-ATPase inthese cells reduces the pool of Src-interacting Na/K-ATPase and thusincreases the basal Src and ERK1/2 activity (25). As depicted in FIG.7A, pNaKtide caused a 10% inhibition of Src in LLC-PK1 cells. However,this inhibition was not statistically significant (p=0.07). On the otherhand, it caused a significant inhibition (over 20%) of basal ERK1/2.This could be explained by that ERK1/2 is a downstream effector of Src.Interestingly, the effect of pNaKtide on Src and ERK in LLC-PK1 cellswas much less than those in TCN23-19 cells (FIG. 7A), showing thatpNaKtide may selectively target the Na/K-ATPase-interacting pool of Src.

To confirm that the effect of pNaKtide on ERK1/2 is due to itsinhibition of Src, the inventors repeated the above experiments inSYF+Src and SYF cells. As shown in FIG. 7B, pNaKtide had no effect onERK1/2 in SYF cells where Src family kinases (Src, Yes, and Fyn) wereknocked out. However, pNaKtide reduced ERK1/2 activity in Src-rescuedSYF cells (SYF+Src).

To test whether the plasma membrane residence makes pNaKtide a relativespecific inhibitor to the Na/K-ATPase-associated Src pool, the inventorsloaded LLC-PK1 cells with NaKtide in the presence of saponin. As shownin FIG. 9—Table 1, NaKtide, like expression of YFP-ND1, caused more than50% inhibition of basal Src activity when loaded by saponin.Interestingly, when the effect of AP-NaKtide on basal Src was measured,like pNaKtide, it barely affected the basal Src activity in LLC-PK1cells.

Second, the inventors measured the effect of pNaKtide in ouabain-inducedactivation of ERK1/2 in LLC-PK1 cells. This showed that 1 μM pNaKtidecompletely abolished ouabain-induced ERK1/2 activation in LLC-PK1 cells(FIG. 8A). To confirm that this is not a cell-specific effect, theinventors repeated the same experiments in primary cultures of cardiacmyocytes. As shown in FIG. 8B, ouabain-induced activation of ERK1/2 wasalso blocked by pNaKtide.

The inventors also compared pNaKtide and PP2, a generic Src inhibitor.As shown in FIG. 9—Table 1, both pNaKtide and PP2 have a similar IC₅₀ onSrc kinase. However, PP2 produced more inhibition on basal Src activitythan that of pNaKtide in both LLC-PK1 and cardiac myocytes. Moreover,when cardiac myocytes were stimulated by IGF, PP2, but not pNaKtide,caused a significant inhibition of ERK1/2 activation (FIG. 8C).Expression of Na/K-ATPase α1 is reduced in some tumor cell lines:Because Src activity is elevated in many types of cancers, the inventorstested whether this elevation is related to the decrease in α1expression. Western blot analyses confirmed that expression of α1 isindeed reduced in some prostate cancer cell lines (i.e., DU145 and PC-3cells), breast cancer cell lines (MCF-7), and all tested neuroblastomacells (FIG. 10A). Immunostaining of Na/K-ATPase α1 also support thisnotion (FIG. 11B). Moreover, knockdown of Na/K-ATPase α1 in vivo,increased basal Src activity in various tissues including liver (FIG. 10B). The inventors also found that the α1 expression was more thandoubled with an increase in cell density from 50% to 90% whereas activeSrc was reduced more than 80% in LLC-PK1 cells (FIG. 10C). However, thisregulation was lost in DU145 cells, apparently because of defects inboth α1 and Src expression (FIG. 10D). When the same experiments wererepeated in LNCaP and PC3 cells, we observed essentially the same defect(data not shown).

pNaKtide inhibits FAK and reduces cell migration: Because Src is theaffector of FAK, a key kinase involved in control of cell migration andthus tumor metastasis, the inventors tested whether pNaKtide can inhibitFAK and tumor cell migration. As depicted in FIG. 11A, pNaKtide caused atime-dependent inhibition of FAK and ERK in TCN23-19 cells where FAK isactivated because of Na/K-ATPase-knockdown-induced Src activation (25).When the effects of pNaKtide were evaluated in DU145 cancer cells,immunostaining with active Src clearly showed a reduction by pNaKtide inboth LNCaP and DU145 cells, but not LLC-PK1 cells (FIG. 11B). When theDU145 cell lysates were analyzed by Western blot, the inventors foundthat pNaKtide reduced not only Src activity in a dose-dependent manner,but also inhibited Src effectors and abolished the expression ofSrc-regulated oncogene, c-Myc (FIG. 11C). Furthermore, the inventorsdiscovered that pNaKtide produced a dose-dependent inhibition of DU145migration (FIG. 12).

Expression of ND1 is sufficient in killing cancer cells where theexpression of Na/K-ATPase is reduced. Some tumor cells express lessNa/K-ATPase and exhibits higher Src activity. The inventors testedwhether expression of a Src-inhibiting Na/K-ATPase fragment (ND1)inhibits the cell growth. As depicted in FIG. 13A and FIG. 13B,expression of YFP-ND1 caused cell growth inhibition or cell death inDU145 and MCF-7 cells.

pNaKtide is effective in inhibiting cell growth in some tumor celllines: The inventors tested whether pNaKtide can be used to inhibittumor cell growth. As shown in FIG. 14, pNaKtide is effective inblocking the growth of several tumor cell lines including prostatecancer, breast cancer and neuroblastoma.

pNaKtide is effective in inhibiting tumor growth in vivo: The inventorsfurther evaluated the effect of pNaKtide in inhibiting tumor growth inNOD/SCID mice. As depicted in FIG. 15A and FIG. 15B, injection ofpNaKtide to NOD/SCID mice bearing xenografted prostate tumors produced adose-dependent inhibition in both the incidence rate as well as thetumor weight. Quantitative measurement of the tumor volume confirmed theeffect of pNaKtide in inhibiting the tumor development is rapid and in adose-dependent manner (FIG. 15C). Administration of 10 mg/kg pNaKtideresulted in over 75% reduction in both tumor volume and actual tumorweight. And apparently, significant reduction of Src activity was foundin the tumors after pNaKtide administration (FIG. 15D).

pNaKtide inhibits angiogenesis: Endothelial cell proliferation is theprerequisite of angiogenesis. As depicted in FIG. 16A, pNaKtideinhibited the proliferation of both HUVEC and HAEC cells in adose-dependent manner. Moreover, the vessel density of tumors fromcontrol mice indicated by CD31 staining was about 10% and pNaKtidetreatment reduced the density to about 2.5% (FIG. 16B and FIG. 16C).When the level of angiogenic factor VEGF was assessed, pNaKtidetreatment produced significant reduction in the tumor homogenates (FIG.16D).

Discussion of the Examples

The inventors herein now show a molecular structure of the Na/K-ATPaseα1 subunit that interacts and inhibits Src. The inventors have alsoengineered a novel peptide Src inhibitor that can target theNa/K-ATPase/Src receptor complex and thus function as an effectiveouabain antagonist in cultured cells.

Identification of NaKtide as a new class of Src inhibitor: The CD3 ofNa/K-ATPaseα1 subunit consists of both N and P domains. The inventorshave shown that CD3 binds the Src kinase domain and inhibits Src kinaseactivity in vitro (24). Based on the newly released crystal structure ofNa/K-ATPase, the N domain is exposed, whereas the P domain is relativelyclose to the membrane (3,5,6).

The inventors now show that the N domain binds and inhibits Src.Interestingly, it is known that the less structural N-terminus of SERCAN domain interacts with phospholamban. The inventors now demonstratethat the ND1 (FIG. 2), the first 50 amino acid residues of the α1 Ndomain, inhibits Src. However, further mapping analyses reveal that thecorresponding phospholamban-binding domain in ND1 (peptide 2, see FIG.4A) actually had no effect on Src kinase activity.

Instead, peptide 3 and peptide 4 showed strong inhibitory effect on Src.Based on the crystal structure as well as NMR data, both peptidespossessed a helix structure, suggesting that helix/helix interactionbetween the ND1 and the Src kinase may be responsible for theNa/K-ATPase-induced Src inhibition. Literature review reveals thatseveral endogenous proteins interact and inhibit Src. Noteworthy areRACK1 and WASP. While RACK1 inhibits Src via its interaction with theSH2 domain (34), WASP does so by binding to the Src kinase domain (22).However, no detailed structural information is available for furthercomparison.

Like inhibitors of other tyrosine kinases, most Src inhibitors are ATPmimetics (35). When the ATP dependence was assessed, the inventors foundthat changes in ATP concentration did not affect NaKtide-induced Srcinhibition. Moreover, it is unlikely that NaKtide acts as a substrateSrc inhibitor (36) since the peptide does not contain Tyr residue.Furthermore, limited structural analyses show that NaKtide interactswith N-lobe, but not the substrate pocket-containing C-lobe, of thekinase domain (Li and Xie, unpublished data). Thus, NaKtide represents anovel class of Src inhibitor.

As a Src inhibitor, both NaKtide and ND1 are potent. The IC₅₀ is closeto 50 nM, comparable to most of reported Src inhibitors. Finally, whenthe effects of NaKtide on other kinases were assessed, the inventorsfound that NaKtide appears to be relatively specific to Src. NaKtideshowed no effect on PKC family of kinases. Its effect on ERK1/2 dependson the expression of Src, indicating that it is not an ERK inhibitor butcan affect ERK signaling by inhibiting Src. This is consistent with thefact that ERK1/2 are well-known effectors of Src kinase. Interestingly,although NaKtide inhibits Lyn, another Src family kinase, it exhibitsmuch lower potency toward Lyn than Src (FIG. 4E).

Development of pNaKtide as a specific ouabain antagonist: Transienttransfection assays indicate that YFP-ND1 resided as a soluble proteinand inhibited basal Src as effective as PP2 (FIG. 3A and FIG. 9—Table1). Moreover, a small fraction of YFP-ND1 was detected in the plasmamembrane. Both FRET and immunoprecipitation assays showed that thisplasma membrane-targeted YFP-ND1 disrupted the interaction between theNa/K-ATPase and Src (FIG. 3B and FIG. 6A). Taken together, thesediscoveries now show that ND1 and its derivative NaKtide may be used asan effective Src inhibitor or a relatively specific ouabain antagonistdepending on its cellular distribution.

This is supported by the experimental evidence where the inventors foundthat loading NaKtide by saponin into LLC-PK1 cells caused about 60%inhibition of basal Src activity as did by PP2 or expression of YFP-ND1.Also, it is demonstrated herein that tagging a positively charged leaderpeptide, such as HIV-Tat to NaKtide, made it readily cell permeable.Moreover, confocal imaging analyses showed that a majority of pNaKtideresided in the plasma membrane and had much less effect on basal Srcactivity in both LLC-PK1 and cardiac myocytes. Interestingly, whenpNaKtide was applied to TCN23-19 cells where the pool of Src-interactingNa/K-ATPase is depleted, in contrast to that in LLC-PK1 cells, pNaKtidecaused about 50% inhibition of cellular Src activity. As such, theplasma membrane-targeted pNaKtide is now believed by the inventorsherein to have a relatively specific effect on theNa/K-ATPase-interacting pool of Src.

The pNaKtide was very effective in blocking the formation ofNa/K-ATPase/Src receptor complex (FIG. 6B). In accordance,ouabain-induced activation of ERK1/2 in LLC-PK1 cells was completelyabolished by 1 μM pNaKtide (FIG. 8A). Moreover, pNaKtide also residedmainly in the plasma membrane in cardiac myocytes and was effective ininhibiting ouabain-induced activation of ERK1/2 (FIG. 8B).

The specificity of pNaKtide toward the Na/K-ATPase/Src complex wasfurther demonstrated by experiments showing that PP2, but not pNaKtide,caused a significant inhibition of IGF-induced ERK1/2 activation (FIG.8C).

Although ouabain and other CTS have been considered only as drugs sincetheir discovery, recent studies have identified both ouabain andmarinobufagenin (MBG) as endogenous steroids whose production andsecretion are regulated by multiple physiological stimuli including ACTHand angiotensin II (37-41). Moreover, they are found to play animportant role in the regulation of renal salt handling, vascular andcardiac contractions (42). Pathologically, they are likely to beinvolved in cardiovascular remodeling seen during chronic renal failureand the pathogenesis of autosomal dominant polycystic kidney disease(ADPKD) by stimulating the proliferation of renal epithelial cells (43).

Therefore, the pNaKtide can be useful in determining the physiology, aswell as to probe the pathological significance, of Na/K-ATPase andendogenous CTS. This is of particular importance since it is nowbelieved that physiologically relevant doses of ouabain and MBG aresufficient to stimulate Src and its down-stream protein kinase cascadesin the heart and kidney (44,45). Moreover, PST 2238, an ouabainantagonist, has been demonstrated as an effective anti-hypertensive drug(44). Therefore, pNaKtide can also be useful as a new therapeutics forcardiovascular diseases where the Na/K-ATPase/Src receptor isover-stimulated.

Identification of key amino acid residues in NaKtide that bind andinhibit Src, where in the kinase domain NaKtide binds to, and how thebinding inhibits Src can be useful, only to reveal the molecularmechanism of Na/K-ATPase-mediated Src regulation, but to assess whetherthis regulation is isoform-specific.

It is to be noted that four different α subunits have been identified.Moreover, Src family kinases include at least nine members.Interestingly, although Src and Lyn are highly homologous, NaKtideappears to be more potent toward Src than Lyn. Thus, it is likely thatthis regulation could be isoform-specific. Also, determining whetherNaKtide affects kinases other than PKC, ERK and Src can be furtheruseful.

The AP-NaKtide and A1N-NaKtide resided mainly in intracellular vesicles.Like pNaKtide, it also had almost no effect on basal Src activity. Assuch, determining whether AP-NaKtide or A1N-NaKtide can blockouabain-induced ERK1/2 activation can also be useful. Moreover,AP-NaKtide and A1N-NaKtide may have a relative specific effect onendocytosis, exocytosis or vesicle recycling since Src is known to playa role in these events. Further, assessment of the ability of pNaKtideas a ouabain antagonist in intact animals or isolated organs can providefurther proof of the usefulness of the novel NaKtide.

Development of pNaKtide as a potential anti-cancer therapeutics: Manytumors have elevated Src activity. Both in vitro and in vivo studieshave demonstrated that cellular Src activity is inversely correlatedwith the amount of Na/K-ATPase. Thus, supplement of Src-inhibitingNa/K-ATPase or its equivalent (ND1 or pNaKtide) may be useful forreducing the tumor growth. Moreover, because Src controls FAK activitythat is required for tumor metastasis, the Na/K-ATPase and itsequivalent may also be effective in preventing tumor metastasis.Consistently, the inventors demonstrate that many tumor cell linesexpress less Na/K-ATPase and have higher Src activity (FIG. 10 and FIG.11). Rescuing these cells with YFP-ND1 or pNaKtide is effective ininhibiting the growth of these tumor cells (FIG. 13 and FIG. 14).Furthermore, pNaKtide inhibits FAK and blocks tumor cell migration invitro (FIG. 11 and FIG. 12). Also, in vivo studies show that IPinjection of pNaKtide can block the growth of xenografted prostate tumorin NOD/SCID mice (FIG. 15). Inhibition of Src in tumors may be involvedin the regulation of angiogenesis by pNaKtide (FIG. 16), which willfurther limit the nutrients supply for the tumor growth. Taken together,these findings indicate that pNaKtide may be useful as an anti-cancertherapeutic agent.

Definitions: The abbreviations used are: A domain, activation domain;CD2, second cytosolic domain; CD3, third cytosolic domain; CTS,cardiotonic steroids; EYFP, enhanced yellow fluorescent protein; ERK,extracellular signal-regulated protein kinase; FAK, focal adhesionkinase; FRET, fluorescence resonance energy transfer; GST,glutathione-S-transferase; IGF-1, insulin-like growth factor 1; MAPK,mitogen-activated protein kinase; N domain, nucleotide-binding domain; Pdomain, phosphorylation domain; PI3K, phosphatidylinositol 3-kinase;PKC, protein kinase C; PLC, phospholipase C;PP2,4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine;SERCA, sarcoplasmic reticulum Ca-ATPase.

SEQUENCE LISTINGS

[SEQ ID NO: 1] peptide 1 . . . MTVAHMWFDNQIHEADTTEN [SEQ ID NO: 2]peptide 2 . . . IHEADTTENQSGVSFDKTSA [SEQ ID NO: 3]peptide 3 . . . SATWLALSRIAGLCNRAVFQ [SEQ ID NO: 4]peptide 4 . . . ALSRIAGLCNRAVFQANQEN [SEQ ID NO: 5]ND1 . . . LTQNRMTVAHMWFDNQIHEADTTENQSGVSFDKTSATW LALSRIAGLCNRAVFQANQEN[SEQ ID NO: 6] pC1 . . . GRKKRRQRRRPPQMTVAHMWFDNQIHEADTTEN[SEQ ID NO: 7] pNaKtide . . . GRKKRRQRRRPPQSATWLALSRIAGLCNRAVFQ[SEQ ID NO: 8] AP-NaKtide. . . RQIKIWFQNRRMKWKKSATWLALSRIAGLCNR AVFQ[SEQ ID NO: 9] A1N-NaKtide . . . KKGKKGKKSATWLALSRIAGLCNRAVFQ

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

REFERENCES

The references discussed above and the following references, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference. Citation of a reference herein shall not beconstrued as an admission that such is prior art to the presentinvention.

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1. A composition of matter comprising an amino acid peptide comprisingat least ten consecutive amino acid residues of the sequenceSATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3], or conservative substitutions ofthe amino acid residues, or substitutions with unnatural amino acids toimprove pharmacodynamic or/and pharmacokinetic properties, wherein thepeptide is capable of binding the kinase domain of Src or Src familykinases.
 2. A composition of claim 1, which further comprises atherapeutically acceptable excipient.
 3. A composition of matter ofclaim 1, wherein the amino acid peptide comprises the sequenceSATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3].
 4. A composition of claim 1,wherein the amino acid peptide comprises a sequence selected from thegroup consisting of: SEQ ID NO: 3; SEQ ID NO: 4; and SEQ ID NO:
 5. 5. Acomposition of claim 1, wherein the amino acid peptide comprises SEQ IDNO: 7 as pNaKtide.
 6. A composition of claim 1, wherein the amino acidpeptide comprises SEQ ID NO: 8 as AP-NaKtide.
 7. A composition of claim1, wherein the amino acid peptide comprises SEQ ID NO: 9 as A1N-NaKtide.8. A nucleic acid sequence encoding a composition of claim
 1. 9. Avector expressing a nucleic acid sequence encoding the peptide of claim5.
 10. A vector expressing a nucleic acid sequence encoding the peptideof claim
 6. 11. A vector expressing a nucleic acid sequence encoding thepeptide of claim
 7. 12. A cell comprising a vector of claim 9 or claim10 or claim
 11. 13. A cell of claim 12, which is E. coli.
 14. A cell ofclaim 12, which is mammalian.
 15. A cell of claim 12, which is a tumorcell.
 16. A monoclonal antibody selective for a composition of claim 1.17. A composition of claim 1, wherein the composition is capable ofaffecting a cellular process selected from the group consisting of:antagonizing a CTS-induced protein kinase cascades; Src inhibition;Na/K-ATPase mimic; Lyn inhibition; ouabain antagonism; FAK/ERK1/2inhibition; anti-angiogenesis and inhibition of cell growth.
 18. Acomposition of claim 1, which is not an ATP analog.
 19. A composition ofclaim 2, which further comprises means to therapeutically permeateplasma membrane.
 20. A composition of claim 2, which further comprisesat least one additional therapeutic composition useful to a treat adisease selected from the group consisting of: cancer; vascular disease;cardiovascular disease; heart disease; bone disease; prostate cancer;breast cancer; neuroblastoma; cardiac hypertrophy; tissue fibrosis;congestive heart failure; ischemia/reperfusion injury and osteoporosis.21. A composition of claim 3, which further comprises a second compoundbound with the amino acid in a location other than SEQ ID NO: 3, whereinthe second compound is selected from the group consisting of:chemotherapeutic drug; toxin; immunological response modifier; enzyme;and radioisotope.
 22. A composition of claim 3, which further comprisesa second compound bound with the amino acid peptide in a location otherthan SEQ ID NO: 3, wherein the second compound is selected from thegroup consisting of: HIV-Tat; Penetratin; and HIV-Tat-S-S; N-terminalsequence of Na/K-ATPase α1; GST; EYFP.
 23. A composition of claim 3,which comprises HIV-Tat-SEQ ID NO:
 3. 24. A composition of claim 3,which comprises a fusion protein, provided that the fusion does notdisrupt the at least ten consecutive residues of SEQ ID NO:
 3. 25. Acomposition of claim 3, wherein the fusion is with GST.
 26. A method tobind a peptide to the kinase domain of Src in a Src-expressing cell,comprising contacting a peptide of claim 1 to at least oneSrc-expressing cell.
 27. A method of claim 26, wherein theSrc-expressing cell is a mammalian cell.
 28. A method of claim 26,wherein the at least one mammalian cell is a cell selected from thegroup consisting of: heart cell, liver cell, vascular cell; breast cell;prostate cell; kidney cell; muscle cell; bone cell; and brain cell. 29.A method of claim 26, wherein the at least one mammalian cell iscultured in vitro.
 30. A method of treating a Src-associated disease ina mammal in need of such treatment, comprising administering atherapeutic composition of claim
 2. 31. A method of claim 30, whereinthe Src-associated disease is selected from the group consisting of:cancer; vascular disease; cardiovascular disease; heart disease; bonedisease; prostate cancer; breast cancer; neuroblastoma; cardiachypertrophy; tissue fibrosis; congestive heart failure;ischemia/reperfusion injury; and osteoporosis.
 32. A method of claim 30,wherein the mammal is a human.
 33. A method of claim 30, wherein thetherapeutic composition comprises an amino acid peptide comprising asequence selected from the group consisting of: SEQ ID NO: 3; SEQ ID NO:4; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO:
 9. 34. Amethod of treating cancer in a mammal in need of such treatment,comprising administering a Src-inhibiting therapeutic composition ofclaim
 2. 35. A method of treating vascular disease in a mammal in needof such treatment, comprising administering a Src-inhibiting therapeuticcomposition of claim
 2. 36. A method of treating heart disease in amammal in need of such treatment, comprising administering aSrc-inhibiting therapeutic composition of claim
 2. 37. A method oftreating prostate cancer in a mammal in need of such treatment,comprising administering a Src-inhibiting therapeutic composition ofclaim
 2. 38. A method of treating breast cancer in a mammal in need ofsuch treatment, comprising administering a Src-inhibiting therapeuticcomposition of claim
 2. 39. A method of treating neuroblastoma in amammal in need of such treatment, comprising administering aSrc-inhibiting therapeutic composition of claim
 2. 40. A method oftreating cardiac hypertrophy in a mammal in need of such treatment,comprising administering a Src-inhibiting therapeutic composition ofclaim
 2. 41. A method of treating tissue fibrosis in a mammal in need ofsuch treatment, comprising administering a Src-inhibiting therapeuticcomposition of claim
 2. 42. A method of treating congestive heartfailure in a mammal in need of such treatment, comprising administeringa Src-inhibiting therapeutic composition of claim
 2. 43. A method oftreating ischemia/reperfusion injury in a mammal in need of suchtreatment, comprising administering a Src-inhibiting therapeuticcomposition of claim
 2. 44. A method of treating osteoporosis in amammal in need of such treatment, comprising administering aSrc-inhibiting therapeutic composition of claim
 2. 45. A method toreduce increased basal Src activity in a tumor cell when the expressionof Na/K-ATPase is reduced, comprising administering a Src-inhibitingcomposition of claim 1 to a Src-expressing tumor cell.
 46. A method toinhibit FAK in a tumor cell comprising administering a Src-inhibitingcomposition of claim 1 to a Src-expressing tumor cell.
 47. A method forscreening at least one test composition to determine whether the atleast one composition affects Src, comprising: introducing a testcomposition comprising a modified amino acid peptide ofSATWLALSRIAGLCNRAVFQ [SEQ ID NO: 3] to Src, wherein the modification isat least one conservative amino acid substitution; and determiningwhether the test composition affects Src.
 48. A method of claim 47,wherein the affect is selected from the group consisting of: Srcbinding; Src inhibition; Src stimulation; Src function; Lyn binding; Lynfunction; Lyn inhibition; ouabain antagonism; Na/K-ATPase function;ERK1/2 function; FAK inhibition.
 49. A method of claim 47, whereinintroducing a test composition is accomplished in vitro.
 50. A method ofclaim 47, wherein introducing a test composition is accomplished in atleast one mammalian cell.
 51. A method of claim 47, wherein introducinga test composition is accomplished in at least one tumor cell line. 52.A method of claim 51, wherein determining whether the compositionaffects Src is measured by cell growth compared to control.
 53. A methodof claim 51, wherein determining whether the composition affects Src ismeasured by cell migration compared to control.
 54. A method of claim51, wherein the at least one mammalian cell is an animal model.
 55. Amethod of claim 51, wherein the animal model is a NOD/SCID mouse.
 56. Anovel Src inhibitor, comprising a composition that targets theNa/K-ATPase/Src receptor complex and antagonizes CTS-induced proteinkinase cascades in one or more cells in need thereof.
 57. A method forinhibiting Src activity or antagonizing CTS-induced signal transductionin a subject in need thereof, comprising administering an effectiveamount of one or more peptides derived from Na/K-ATPase or abiologically active fragments thereof.
 58. A composition that functionsas an effective Src inhibitor, is not an ATP analog, does not directlyaffect PKC and ERK families of serine/threonine kinases, and inhibitsLyn, a Src family tyrosine kinase, comprising one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof.
 59. Ahighly positively charged leader peptide conjugate comprisingHIV-Tat-NaKtide (pNaKtide) which readily enters cells and resides in theplasma membrane.
 60. A highly positively charged leader peptideconjugate comprising Penetratin-NaKtide (AP-NaKtide) which readilyenters cells and resides in vesicles.
 61. A highly positively chargedleader peptide conjugate comprising HIV-Tat-SS-NaKtide (ssNaKtide) whichreadily enters cells and resides in the cytosol.
 62. A highly positivelycharged leader peptide conjugate comprising Na/K-ATPase α1N-terminus-NaKtide (A1N-NaKtide) which readily enters cells and residesin cytosol.
 63. A method for targeting the Na/K-ATPase-interacting poolof Src and acting as a potent ouabain antagonist in one or more cells inneed thereof, comprising administering an effective amount of pNaKtide.64. A method for targeting vesicular Src and acting as a potent ouabainantagonist in one or more cells in need thereof, comprisingadministering an effective amount of AP-NaKtide.
 65. A method fortargeting whole cell Src and acting as a potent ouabain antagonist inone or more cells in need thereof, comprising administering an effectiveamount of ssNaKtide.
 66. A method for targeting vesicular Src and actingas a potent ouabain antagonist in one or more cells in need thereof,comprising administering an effective amount of A1N-NaKtide.
 67. Amethod for disrupting the formation of Na/K-ATPase/Src receptor complexin a dose-dependent manner, comprising administering an effective amountof one or more peptides derived from Na/K-ATPase or biologically activefragments thereof.
 68. A method for blocking ouabain-induced activationof Src and a down-stream signaling pathway, such as ERK1/2, in a subjectin need thereof, comprising administering an effective amount of one ormore peptides derived from Na/K-ATPase or biologically active fragmentsthereof.
 69. Use of one or more peptides derived from Na/K-ATPase orbiologically active fragments thereof as a ouabain antagonist.
 70. Amethod for determining the physiological and pathological significanceof the signaling function of Na/K-ATPase and CTS, comprising using oneor more peptides derived from Na/K-ATPase or biologically activefragments thereof as a probe.
 71. A Src inhibitor comprising acomposition capable of targeting the plasma membrane Na/K-ATPase/Srccomplex selected from one or more of SEQ ID NOs: 1, 2, 3, 4 and 5, orbiologically active fragments thereof.
 72. A composition capable ofselectively targeting the Na/K-ATPase-interacting pool of Src and offunctioning as an effective ouabain antagonist in one or more cells inneed thereof, comprising one or more peptides derived from Na/K-ATPaseor biologically active fragments thereof.
 73. As a Src inhibitor that isspecific to Src and shows no direct effect on PKC family of kinases,comprising one or more of ND1, NaKtide, pNaKtide, AP-NaKtide, ssNaKtide,A1N-NaKtide, and biologically active fragments thereof.
 74. A specificouabain antagonist, comprising one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof tagged with apositively charged leader peptide.
 75. The antagonist of claim 74,wherein the tagged peptide comprises HIV-Tat or Penetratin, HIV-Tat-SS(linked to NaKtide via SS bond), or Na/K-ATPase N-terminus tagged toNaKtide.
 76. A method for determining the physiology and/or probing thepathological significance of Na/K-ATPase and endogenous CTS, comprisingusing one or more peptides derived from Na/K-ATPase or biologicallyactive fragments thereof.
 77. A therapeutic composition forcardiovascular diseases where the Na/K-ATPase/Src receptor isover-stimulated, comprising one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof.
 78. A method ofinducing cell growth inhibition and/or cell death in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a non ATP-competitive inhibitor of Src and a CTSantagonist.
 79. A composition for preventing CTS-provoked signalingpathway in a subject in need thereof, the composition comprising one ormore peptides derived from Na/K-ATPase or biologically active fragmentsthereof.
 80. A method for substantially abolishing ouabain-provokedsignaling transduction in the heart in a subject in need thereof,comprising administering an effective amount of one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof. 81.The use of one or more peptides derived from Na/K-ATPase or biologicallyactive fragments thereof, in the preparation of a medicament for thetreatment of a cancer related disorder or a cardiac disease relateddisorder.
 82. A pharmaceutical composition comprising one or morepeptides derived from Na/K-ATPase or biologically active fragmentsthereof, and a physiologically acceptable carrier.
 83. The compositionof claim 82, wherein the composition is adapted for use as a treatmentfor cardiac hypertrophy, tissue fibrosis and/or congestive heartfailure.
 84. The composition of claim 82, wherein the composition isadapted for use as a chemotherapeutic agent.
 85. The composition ofclaim 82, wherein the composition is adapted for use as a treatment fora cancer related disorder.
 86. The composition of claim 82, wherein thecomposition is adapted for use as a treatment for a cancer relateddisorder selected from one or more of prostate cancer, breast cancer,and neuroblastoma.
 87. A method of identifying a candidate compound forthe treatment of a disorder associated with one or more of cardiachypertrophy, tissue fibrosis, congestive heart failure or cancer, themethod comprising: providing an assay for detecting an interactionbetween Na/K-ATPase and Src which inhibits Src activity; conducting theassay with a test compound; and identifying a test compound that is anon ATP-competitive Src inhibitor and a CTS antagonist, wherein a testcompound that significantly inhibits the disorder is a candidatecompound for the treatment of the disorder.
 88. A method of identifyinga candidate compound for the treatment of a disorder associated with oneor more of cardiac hypertrophy, tissue fibrosis, congestive heartfailure or cancer, the method comprising: providing a model of thedisorder; contacting the model with a test compound; detecting a levelof: i) one or more peptides derived from Na/K-ATPase or biologicallyactive fragments and active Src thereof, ii) or Na/K-ATPase or CTSbinding; and comparing the level of the peptides and Src to a reference,wherein a test compound that causes a significant difference in a levelof the peptides and active Src as compared to the reference is acandidate compound for the treatment of the disorder.
 89. A method ofdiagnosing a subject with a disorder associated with one or more ofcardiac hypertrophy, tissue fibrosis, congestive heart failure orcancer, the method comprising: providing a sample from the subject;detecting in the sample a level of: i) one or more peptides derived fromNa/K-ATPase or biologically active fragments and active Src thereof, ii)or Na/K-ATPase or CTS binding; and comparing the level of peptides andactive Src to a reference, wherein a significant difference in a levelof the peptides as compared to the reference indicates that the subjecthas the disorder.
 90. A method of evaluating a treatment for a disorderassociated with one or more of cardiac hypertrophy, tissue fibrosis,congestive heart failure or cancer, the method comprising: providing asample from the subject; detecting in the sample a level of: i) one ormore peptides derived from Na/K-ATPase or biologically active fragmentsand active Src thereof, ii) or Na/K-ATPase or CTS binding; andadministering one or more doses of a treatment, and comparing the levelof the peptides to a reference, wherein a significant difference in alevel of the peptides and Src, as compared to an unaffected individual,as compared to the reference indicates the efficacy of the treatment.91. The method of claim 90, wherein the reference represents a level ofthe peptides and Src prior to administration of the treatment.
 92. Themethod of claim 90 wherein the sample is from cardiac tissue or a cancercell of the subject.
 93. A method of determining a subject's risk fordevelopment of a complication of a disorder associated with one or moreof cardiac hypertrophy, tissue fibrosis, congestive heart failure orcancer, the method comprising: providing a sample from the subject;detecting in the sample a level of: i) one or more peptides derived fromNa/K-ATPase or biologically active fragments and active Src thereof, ii)or Na/K-ATPase or CTS binding; and comparing the level of the peptidesand Src to a reference, wherein a significant difference in a level ofthe peptides as compared to the reference indicates the subject's riskof developing the complication.
 94. A method of determining when atreatment modality administered to a subject to treat a disorderassociated with one or more of cardiac hypertrophy, tissue fibrosisand/or congestive heart failure can be stopped, the method comprising:providing a sample from the subject; detecting in the sample a level of:i) one or more peptides derived from Na/K-ATPase or biologically activefragments and active Src thereof, ii) or Na/K-ATPase or CTS binding; andcomparing the level of the peptides and Src to a reference, wherein alevel of the peptides and Src that approaches the level of the peptidesand Src in the reference indicates whether the treatment can be stopped.95. A method of determining when a treatment for a disorder associatedwith one or more of cardiac hypertrophy, tissue fibrosis, congestiveheart failure or cancer, should be initiated in a subject, the methodcomprising: providing a sample from the subject; detecting in the samplea level of: i) one or more peptides derived from Na/K-ATPase orbiologically active fragments and active Src thereof, ii) or Na/K-ATPaseor CTS binding; and comparing the level of to a reference, wherein asignificant difference in a level of the peptides and active Src ascompared to the reference indicates whether the treatment should beinitiated.
 96. The method of claim 95, wherein the reference representsa level of peptides and active Src in an unaffected subject.
 97. Amethod for preventing or treating a condition mediated by a ouabainsteroid in a subject, comprising administering one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof,and/or an agonist or antagonist thereof.
 98. The method of claim 97,wherein the condition is one or more of cancer, cardiac hypertrophy,tissue fibrosis or congestive heart failure.
 99. A method foridentifying one or more of: i) a substance that modulates a ouabainsteroid receptor, a Na/K-ATPase receptor and/or a Na/K-ATPase/Srcreceptor complex, ii) a process mediated by a ouabain steroid receptor,a Na/K-ATPase receptor and/or a Na/K-ATPase/Src receptor complex, iii)degradation of a ouabain steroid receptor, a Na/K-ATPase receptor and/ora Na/K-ATPase/Src receptor complex, iv) a ouabain steroid receptorand/or Na/K-ATPase receptor signaling transduction pathway, v) acondition mediated by a ouabain steroid receptor, a Na/K-ATPase receptorand/or a Na/K-ATPase/Src receptor complex, vi) a steroid receptortransactivation, and/or inhibits or potentiates the interaction of aouabain steroid receptor, a Na/K-ATPase receptor and/or aNa/K-ATPase/Src receptor complex, comprising assaying for a substancethat inhibits or stimulates a ouabain steroid receptor, Na/K-ATPasereceptor and/or a Na/K-ATPase receptor/Src complex.
 100. A method forevaluating a substance, comprising: reacting one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof and areceptor therefore with a test substance, wherein the peptides andreceptor bind to form a complex; and comparing to a control in theabsence of the test substance to determine if the substance stimulatesor inhibits the binding of the peptides to the receptor.
 101. A methodof conducting a drug discovery business comprising: a) providing amethod for identifying a substance as claimed in claim 100; b)conducting therapeutic profiling of substances identified in step a), orfurther analogs thereof, for efficacy and toxicity in animals; and c)formulating a pharmaceutical preparation including one or moresubstances identified in step b) as having an acceptable therapeuticprofile.
 102. A method for regulating the Na/K-ATPase/Src receptorcomplex in a subject comprising one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof, a complex thereof;or interaction thereof with a receptor.
 103. Antibodies specific forpeptides derived from Na/K-ATPase or biologically active fragmentsthereof.
 104. Antibodies labeled with a detectable substance and used todetect proteins or complexes peptides derived from Na/K-ATPase orbiologically active fragments thereof in biological samples, tissues,and cells.
 105. Antibodies having uses in therapeutic applications, andin conjugates and immunotoxins as target selective carriers of variousagents which have therapeutic effects including chemotherapeutic drugs,toxins, immunological response modifiers, enzymes, and radioisotopes.106. A pharmaceutical composition adapted for administration to asubject for the prevention or treatment of a condition mediated by asteroid receptor comprising an effective amount of one or more peptidesderived from Na/K-ATPase or biologically active fragments thereof, oragonists or antagonists thereof, or an agent, compound or substanceidentified using a method of claim 99 and a pharmaceutically acceptablecarrier, diluent or excipient.
 107. A pharmaceutical compositioncomprising an effective amount of one or more peptides derived fromNa/K-ATPase or biologically active fragments thereof or an agonist orantagonist thereof, and an appropriate carrier, diluent, or excipient.108. A pharmaceutical composition adapted for administration to asubject for the prevention or treatment of a condition mediated by asteroid receptor, in particular a condition mediated by a ouabainreceptor, and an appropriate carrier, diluent, or excipient.
 109. Use ofpeptides derived from Na/K-ATPase, biologically active fragments thereofor agonists or antagonists thereof, for the manufacture of, or in thepreparation of a medicament.